Process technology for biological product manufacturing and downstream purification

ABSTRACT

Provided herein are, inter alia, biological manufacturing and downstream purification processes.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.17/474,232, filed Sep. 14, 2021, which claims the benefit of priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/077,766,filed on Sep. 14, 2020, Provisional Application No. 63/154,108, filed onFeb. 26, 2021, and Provisional Application No. 63/154,109, filed on Feb.26, 2021, the entire contents of each of which is incorporated herein byreference in their entireties.

FIELD OF INVENTION

New processes and methods for manufacturing and downstream purificationof biological products are provided.

BRIEF SUMMARY

Provided herein, inter alia, are processes and apparatuses for purifyinga biological product. In aspects, provided herein is a process forpurifying a biological product, where the process includes receiving,via an input line, a heterogeneous mixture containing the biologicalproduct, removing impurities from the heterogeneous mixture byfiltration in a dynamic filtration module. Impurities are removed fromthe heterogeneous mixture by feeding the biological product from atleast one output head in fluid communication with the input line to thedynamic filtration module under negative pressure, thereby producing afiltrate comprising the biological product.

The dynamic filtration module includes a dynamic filtration apparatus, atarget region that is configured to receive the heterogeneous mixturefrom at least one output head, and a membrane support member with asubstantially smooth contact surface that is in communication with avacuum collection system that is positioned between the feed reel andthe collection reel. Additionally dynamic filtration apparatus includesa filter membrane extending between a feed reel and a collection reelwith at least one support member having a substantially smooth contactsurface. Purifying the biological product further includes transferringthe filtrate to a first module capable of separating the solution intotwo or more fractions, where at least one fraction contains thebiological product; the first module includes an affinity-basedpurification apparatus. The affinity-based purification apparatus has atleast one first inlet and at least one first outlet configured to permitfluid flow between the at least one first inlet and the at least onefirst outlet via a mechanical rotary system. The mechanical rotarysystem includes a vessel carousel containing at least one discretevessel comprising a suspension of beads. As described herein, theprocess further includes transferring the fraction containing thebiological product from the at least one outlet of the first module to asecond module having at least one inlet for receiving flow from the atleast one first outlet of the first module. The second module includesat least one free-flow electrophoresis apparatus, and the second modulehas at least one second inlet and at least one second outlet and isconfigured to permit continuous fluid flow between the second inlet andthe second outlet; and thereby recovering the biological product.

In embodiments, the affinity-based purification apparatus furtherincludes a lid system and a collection vessel system in fluidcommunication with the at least one discrete vessel. For example, thelid system has at least one lid having a gasket, at least two bufferinlets, a filling inlet, a gas inlet, and a venting valve. Moreover, thelid system is capable of motion along the z-axis. The vessel carousel ofthe affinity-based purification apparatus is capable of rotationalmotion in a plane transverse to the z-axis, and the collection vessel iscapable of motion along the z-axis.

As described herein, the vessel carousel of the affinity-basedpurification method includes at least one position to bind thebiological product, at least one position to wash to remove unboundproducts, at least one position to elute and collect the biologicalproduct, and at least one regeneration position to enable recycling ofthe beads.

In examples, the surface of the beads of the affinity-based purificationis coupled to Protein A, Protein G, Protein L, an antigenic protein, aprotein, a receptor, an antibody, or an aptamer configured toselectively bind said biological product. The initial concentration ofthe beads (e.g., in the discrete vessel at the position to bind thebiological product) is in a concentration range from about 0.01% toabout 25% by weight. Alternatively, the initial concentration of beadsis in the range from about 0.01% to about 20%, or from about 0.01% toabout 10%, or from about 0.01% to about 5%, or from about 1% to about20%, or from about 5% to about 10% by weight). In examples, the beadshave a diameter ranging from about 0.2 μm to about 200 μm. In otherexamples, the beads have a diameter from about 0.2 μm to about 100 μM,or from about 1 μm to about 200 μm, or from about 10 μm to about 200 μm,or from about 20 μm to about 200 μm, or from about 30 μm to about 200μm, or from about 50 to about 200 μm, or from about 150 to about 200 μm.Alternatively, the beads have a diameter from about 1 μm to about 100μm, or from about 50 μm to about 100 μm.

In embodiments, the beads (e.g., of the affinity-based purification)remain mobile during the process to maintain an increased surface areaavailable for binding. For example, the beads remain separated(circulating or dispersed) in solution during the process (e.g., theyare discrete beads). Moreover, the mobile beads may also mean that thebeads do not aggregate together, e.g., at least two or more beadsaggregated or grouped together. Additionally, the mobile beads may alsomean that the beads may form small aggregates that remain dispersed andfree to move within a solution. Conversely the beads used herein are notpacked, but remain mobile, and free to move within a solution.

In embodiments, the free-flow electrophoresis apparatus includeselectrode channels, including an anodic electrode channel and a cathodicelectrode channel, having liquid contact with a main separation channelvia a wall gap.

The free-flow electrophoresis apparatus has at least one electrodechannel de-bubbler including at least one gas permeable and hydrophobicmembrane configured to remove bubbles by a vacuum system creating abubble-free main separation channel, and at least one liquid circuitbreaker. In embodiments, the at least one de-bubbler of the free-flowelectrophoresis apparatus is configured to continuously remove O₂ and H₂gas bubbles that evolve in the electrode channels under applied voltage.In some embodiments, removal of electrolysis bubbles from the electrodechannels is essential to enable continuous operation for substantiallylong periods of time. In examples, the de-bubbler system utilizes ahydrophobic PTFE membrane to create a water-tight seal atop theelectrode channel that permits continuous removal of electrolysisbubbles at the point of generation by exposure to a vacuum system. Inexamples, the vacuum gauge pressure ranges from about −0.05 bar to about−0.4 bar. Contrary to current methods, the process described hereinremoves gas bubbles prior to entering the main separation channel.

In embodiments, the liquid circuit breaker of the free-flowelectrophoresis apparatus includes a pressurized vessel configured tomaintain flow rate and creates droplets that break the circuit from thesolution connected to voltage.

In embodiments, the purification process maintains approximately aconstant flow rate in the dynamic filtration module, the first module,and the second module. For example, the flow rate ranges from about 0.1mL/minute to about 50 mL/minute, or from about 5 mL/minute to about 10mL/minute.

In embodiments, the process for purifying a biological product isperformed at a temperature in the range of about 4° C. to about 37° C.

In further embodiments, the processes may include at least two dynamicfiltration modules, where each dynamic filtration module has a filtermembrane includes the same or different pore sizes (e.g., wherein theheterogeneous mixture contacts a larger pore size filter membrane first(e.g., 0.45 μm), followed by contact with a smaller pore size filtermembrane next (e.g., 0.2 μm).

In embodiments, the process includes at least two free-flowelectrophoresis modules configured to operate in an isoelectric focusingmode, a zone electrophoresis mode, an isotachophoresis mode, orcombinations thereof. The processes described herein further includes atleast two dynamic filtration modules, at least two affinity-basedpurification modules, or at least two free-flow electrophoresis modulesoperated in parallel.

In aspects, provided herein is a dynamic filtration apparatus forremoving impurities from a biological product in a heterogeneousmixture. The apparatus includes a filter membrane extending between afeed reel and a collection reel, the filter membrane having a targetregion that is configured to receive the heterogeneous mixture from atleast one output head configured to dispense the heterogeneous mixtureonto the target region. The membrane support structure of the apparatushas a substantially smooth contact surface to structurally support aportion of the filter membrane that is positioned between the feed reeland the collection reel to create the target region. Moreover, thedynamic filtration apparatus has at least one support member with asubstantially smooth contact surface to stabilize the transport of thefilter membrane across the membrane support structure. The dynamicfiltration apparatus has a system configured to control the transportvelocity of the filter membrane. The dynamic filtration apparatus has avacuum system having at least one vacuum line in communication with themembrane support structure and configured to apply negative gaugepressure across the dynamic filter membrane, where the negative pressureenables collection of the filtrate containing the biological product. Inother examples, the dynamic filtration apparatus includes a wash bufferline.

In embodiments, the dynamic filtration apparatus has a filter membranewhich may include polyethersulfone (PES), hydrophilic polysulfone,cellulose ester, cellulose acetate, polyvinylidene fluoride (PVDF),hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE),hydrophilic PTFE, or any combination thereof. The pore size of thefilter membrane is in the range from about 0.1 μm to about 1 μm. Inother examples, the pore size is in the range from about 0.1 to about0.9 μm, or from about 0.1 μm to about 0.8 μm, or from about 0.1 μm toabout 0.7 μm, or from about 0.1 μm to about 0.6 μm, or from about 0.1 μmto about 0.5 μm, or from about 0.1 μm to about 0.4 μm, or from about 0.1μm to about 0.3 μm, or from about 0.1 μm to about 0.2 μm. As describedherein, if two or more dynamic filtration apparatuses are used, they mayinclude filter membranes of similar or different sizes.

The dynamic filtration apparatus described herein includes a membranesupport structure that has a series of parallel slots, e.g., from about1 to about 10 parallel slots. In specific examples, the membrane supportstructure has 5 parallel slots.

The dynamic filtration apparatus described herein includes a membranesupport structure with a substantially smooth contact surface, where thecontact surface is a measure of its static coefficient of friction,e.g., from about 0.01 to about 0.1, or from about 0.01 to about 0.05, orfrom about 0.05 to about 0.1. In specific examples, the staticcoefficient of friction is 0.04.

In embodiments, the vacuum system of the dynamic filtration module isconfigured to apply negative gauge pressure, e.g., in the range fromabout t −0.05 bar to about −0.98 bar.

In aspects, provided herein is a free-flow electrophoresis apparatus forseparating a mixture into two or more fractions, at least one fractioncontaining a biological product. The free-flow electrophoresis apparatusincludes at least one inlet and at least one outlet configured to permitcontinuous fluid flow between the at least one inlet and the at leastone outlet; at least one fluidic channel created between two parallelplates and configured to create an electric field gradient orthogonal tothe direction of fluid flow; electrode channels comprising an anodicelectrode channel and a cathodic electrode channel, wherein theelectrode channels are configured to be connected to the main separationchannel by liquid contact through a wall gap positioned between theelectrode channels and the main separation channel; at least oneelectrode channel de-bubbler comprising at least one gas permeable andhydrophobic membrane or porous material configured to removeelectrolysis bubbles near the point of generation by a vacuum system tocreate a bubble-free main separation channel; at least one liquidcircuit breaker configured to disconnect the solution connected tovoltage prior to interacting with at least one sensor or detector; anactive cooling system; and at least one collection vessel.

The free-flow electrophoresis apparatus described herein provides for anelectrode channel having a de-bubbler wherein the top portion of theelectrode channels are sealed with at least one gas permeable andhydrophobic membrane in communication with a vacuum system to removebubbles, and wherein the electrode channels are open at the bottom ofthe channels and configured to enable liquid contact of electrodesolution with the main separation channel solution through a wall gap.

The free-flow electrophoresis apparatus has at least one electrodechannel de-bubbler including at least one gas permeable and hydrophobicmembrane configured to remove bubbles by a vacuum system creating abubble-free main separation channel, and at least one liquid circuitbreaker.

In embodiments, the free-flow electrophoresis apparatus furthercomprises at least one de-bubbler system to continuously remove O₂ andH₂ gas bubbles that evolve in the electrode channels under appliedvoltage. In some embodiments, removal of electrolysis bubbles isessential to enable continuous operation for substantially long periodsof time. In examples, the de-bubbler system utilizes a hydrophobic PTFEmembrane to create a water-tight seal atop the electrode channel thatpermits continuous removal of electrolysis bubbles at the point ofgeneration by exposure to a vacuum system. In examples, the vacuum gaugepressure ranges from about −0.05 bar to about −0.4 bar. Contrary tocurrent methods, the process described herein removes gas bubbles priorto entering the main separation channel.

In embodiments, the wall gap (e.g., the space where the electrodechannels are open at the bottom of the channels and are configured toenable liquid contact of electrode solution with the main separationchannel solution) is about 0.01 mm to about 0.25 mm. In examples, thewall gap is from about 0.01 mm to about 0.2 mm, or from about 0.01 mm toabout 0.015 mm, or from about 0.01 mm to about 0.01 mm.

In other embodiments, the liquid circuit breaker of the free-flowelectrophoresis apparatus includes a pressurized vessel configured tomaintain flow rate and creates droplets that break the circuit from thesolution connected to voltage.

In embodiments, the free-flow electrophoresis apparatus further includesan in-line sensor. In examples, the in-line sensor may include a flowsensor, a pH sensor, a conductivity sensor, or any combination thereof.

In embodiments, the free-flow electrophoresis apparatus described hereincan include at least two free-flow electrophoresis apparatuses connectedin series and operated in an isoelectric focusing mode, a zoneelectrophoresis mode, an isotachophoresis mode, or combinations thereof,to enable a staged purification.

Also provided herein, is the use of the free-flow electrophoresisapparatus to purify a biological product from a mixture. The disclosurefurther provides use of the dynamic filtration apparatus to purify abiological product from a heterogeneous mixture.

In aspects, provided herein is a process for purifying a biologicalproduct. The process comprises receiving, via an input line, aheterogeneous mixture containing the biological product. In embodiments,the process comprises continuously receiving, via an input line, aheterogeneous mixture containing the biological product. In embodiments,the biological product includes a protein or fragment thereof (apolypeptide), an antibody or fragment thereof, a cytokine, a chemokine,a growth factor, an enzyme, an oligonucleotide, a virus, an adenovirus,an adeno-associated virus (AAV), or a lentivirus.

In embodiments, the process includes removing impurities (e.g., largeimpurities such as cells, cell debris, and aggregates) from theheterogeneous mixture by dynamic filtration. In some embodiments, thedynamic filtration process may be a continuous process for removinglarge impurities from a heterogeneous mixture. Said dynamic filtrationprocess includes at least one dynamic filtration module thatcontinuously feeds the heterogeneous mixture containing the biologicalproduct from at least one output head in fluid communication with theinput line to the dynamic filtration module under negative pressure,thereby producing a filtrate comprising the biological product.

In embodiments, the process includes transferring the filtrate to afirst module capable of separating the solution into two or morefractions, wherein at least one fraction contains the biologicalproduct. In other embodiments, the process includes continuouslytransferring the filtrate to a first module capable of separating thesolution into two or more fractions, wherein at least one fractioncontains the biological product. For example, separating the solutioninto two or more fractions, may include one fraction containing thebiological product, and the at least one other fraction containing smallimpurities (e.g., host cell proteins, undesired proteins and peptides,undesired antibodies, undesired nucleic acids and oligonucleotides,viruses, salts, buffer components, surfactants, sugars, metalliccontaminants, leachables, media components, and/or naturally-occurringorganic molecules with which it is naturally associated).

In embodiments, the first module comprises an affinity-based, magneticpurification apparatus. In examples, the first module has at least onefirst inlet and at least one first outlet and is configured to permitcontinuous fluid flow between the first inlet and the first outlet via aloop conveyor system. In other examples, the first module has at leastone first inlet and at least one first outlet and is configured topermit continuous fluid flow between the first inlet and the firstoutlet via a pick and place robotics system.

In embodiments, the process includes transferring the fractioncontaining the biological product from the at least one first outlet ofthe first module to a second module having at least one inlet forreceiving flow from the at least one first outlet of the first module,and the second module comprises a charge-based, magnetic purificationapparatus or an isoelectric point-based, fluidic purification apparatus(also referred to herein as a free-flow electrophoresis apparatus). Inother embodiments, the process includes continuously transferring thefraction containing the biological product from the at least one firstoutlet of the first module to a second module having at least one inletfor receiving flow from the at least one first outlet of the firstmodule, and the second module comprises a charge-based, magneticpurification apparatus or an isoelectric point-based, fluidicpurification apparatus, also referred to herein as a free-flowelectrophoresis apparatus. In examples, the second module comprises acharge-based, magnetic purification apparatus having at least one secondinlet and at least one second outlet and is configured to permitcontinuous fluid flow between the second inlet and the second outlet viaa loop conveyor system. In some examples, the second module comprises acharge-based, magnetic purification apparatus having at least one secondinlet and at least one second outlet and is configured to permitcontinuous fluid flow between the second inlet and the second outlet viaa pick and place robotics system. In other examples, the second modulecomprises a free-flow electrophoresis apparatus having at least onesecond inlet and at least one second outlet and is configured to permitcontinuous fluid flow between the second inlet and the second outlet. Inembodiments, the process described herein thereby purifies thebiological product.

In embodiments, also provided herein is a process for purifying abiological product including continuously receiving, via an input line,a heterogeneous mixture containing the biological product, and removinglarge impurities from the heterogeneous mixture by dynamic filtration.In some embodiments, the dynamic filtration process may be a continuousprocess for removing large impurities from a heterogeneous mixture. Saiddynamic filtration process includes a dynamic filtration module thatcontinuously feeds the biological product from at least one output headin fluid communication with the input line to the dynamic filtrationmodule under negative pressure, thereby producing a filtrate comprisingthe biological product.

In embodiments, the process includes transferring the filtrate to afirst module capable of separating the solution into two or more,wherein at least one fraction contains the biological product. In otherembodiments, the process includes continuously transferring the filtrateto a first module capable of separating the solution into two or morefractions, wherein at least one fraction contains the biologicalproduct. In examples, the first module includes an affinity-basedpurification apparatus. In examples, the first module has at least onefirst inlet and at least one first outlet and is configured to permitcontinuous fluid flow between the first inlet and the first outlet via amechanical rotary system. In other examples, the first module has atleast one first inlet and at least one first outlet and is configured topermit continuous fluid flow between the first inlet and the firstoutlet via a staged linear system.

In embodiments, the process includes transferring the fractioncontaining the biological product from the at least one first outlet ofthe first module to a second module having at least one inlet forreceiving flow from the at least one first outlet of the first module,and the second module includes a charge-based purification apparatus oran isoelectric point-based, fluidic purification apparatus, alsoreferred to herein as a free-flow electrophoresis apparatus. In otherembodiments, the process includes continuously transferring the fractioncontaining the biological product from the at least one first outlet ofthe first module to a second module having at least one inlet forreceiving flow from the at least one first outlet of the first module,and the second module includes a charge-based purification apparatus oran isoelectric point-based, fluidic purification apparatus, alsoreferred to herein as a free-flow electrophoresis apparatus. Inexamples, the second module comprises a charge-based purificationapparatus having at least one second inlet and at least one secondoutlet and is configured to permit continuous fluid flow between thesecond inlet and the second outlet via a mechanical rotary system. Insome examples, the second module has at least one second inlet and atleast one second outlet and is configured to permit continuous fluidflow between the second inlet and the second outlet via a staged linearsystem. In other examples, the second module comprises a free-flowelectrophoresis apparatus having at least one second inlet and at leastone second outlet and is configured to permit continuous fluid flowbetween the second inlet and the second outlet. In embodiments, theprocess described herein thereby purifies the biological product.

In embodiments, also provided herein is a process for purifying abiological product including continuously receiving, via an input line,a heterogeneous mixture containing the biological product, and removinglarge impurities from the heterogeneous mixture by dynamic filtration.In some embodiments, the dynamic filtration process may be a continuousprocess for removing large impurities from a heterogeneous mixture. Saiddynamic filtration process includes a dynamic filtration module thatcontinuously feeds the biological product from at least one output headin fluid communication with the input line to the dynamic filtrationmodule under negative pressure, thereby producing a filtrate comprisingthe biological product.

In embodiments, the process includes transferring the filtrate to afirst module capable of separating the solution into two or morefractions wherein at least one fraction contains the biological product.In other embodiments, the process includes continuously transferring thefiltrate to a first module capable of separating the solution into twoor more fractions, wherein at least one fraction contains the biologicalproduct. In embodiments, the first module includes an affinity-based,fluidic purification apparatus. In examples, the first module has atleast one first inlet and at least one first outlet and is configured topermit continuous fluid flow between the first inlet and the firstoutlet.

In embodiments, the process includes transferring the fractioncontaining the biological product from the at least one first outlet ofthe first module to a second module having at least one inlet forreceiving flow from the at least one first outlet of the first module,and the second module includes a charge-based, fluidic purificationapparatus or an isoelectric point-based, fluidic purification apparatus,also referred to herein as a free-flow electrophoresis apparatus. Inembodiments, the process includes continuously transferring the fractioncontaining the biological product from the at least one first outlet ofthe first module to a second module having at least one inlet forreceiving flow from the at least one first outlet of the first module,and the second module includes a charge-based, fluidic purificationapparatus or an isoelectric point-based, fluidic purification apparatus,also referred to herein as a free-flow electrophoresis apparatus. Inexamples, the second module comprises a charge-based, fluidicpurification apparatus having at least one second inlet and at least onesecond outlet and is configured to permit continuous fluid flow betweenthe second inlet and the second outlet. In other examples, the secondmodule comprises a free-flow electrophoresis apparatus having at leastone second inlet and at least one second outlet and is configured topermit continuous fluid flow between the second inlet and the secondoutlet. In embodiments, the process described herein thereby purifiesthe biological product.

In embodiments, the process includes transferring the filtrate to afirst module capable of separating the solution into two or morefractions, wherein at least one fraction contains the biologicalproduct. In other embodiments, the process includes continuouslytransferring the filtrate to a first module capable of separating thesolution into two or more fractions, wherein at least one fractioncontains the biological product. In embodiments, the first modulecomprises an affinity-based tangential flow filtration (TFF)purification apparatus. In examples, the first module has at least onefirst inlet and at least one first outlet and is configured to permitcontinuous fluid flow between the first inlet and the first outlet.

In embodiments, the process includes transferring the fractioncontaining the biological product from the at least one first outlet ofthe first module to a second module having at least one inlet forreceiving flow from the at least one first outlet of the first module,and the second module comprises a charge-based TFF purificationapparatus or an isoelectric point-based, fluidic purification apparatus,also referred to herein as a free-flow electrophoresis apparatus. Inother embodiments, the process includes continuously transferring thefraction containing the biological product from the at least one firstoutlet of the first module to a second module having at least one inletfor receiving flow from the at least one first outlet of the firstmodule, and the second module comprises a charge-based TFF purificationapparatus or an isoelectric point-based, fluidic purification apparatus,also referred to herein as a free-flow electrophoresis apparatus. Inexamples, the second module comprises a charge-based TFF purificationapparatus having at least one second inlet and at least one secondoutlet and is configured to permit continuous fluid flow between thesecond inlet and the second outlet. In other examples, the second modulecomprises a free-flow electrophoresis apparatus having at least onesecond inlet and at least one second outlet and is configured to permitcontinuous fluid flow between the second inlet and the second outlet. Inembodiments, the process described herein thereby purifies thebiological product.

As described herein, the process of removing large impurities from theheterogeneous mixture does not include centrifugation, disk-stackcentrifugation, depth filtration, static filtration, tangential flowfiltration, or any combination thereof. Alternatively, the processdescribed herein may receive a heterogeneous mixture containing abiological product via an input line derived from any large impurityremoval input, for example, without intent to be limiting, a centrifugeand depth filtration process.

As described herein, the process of continuously removing largeimpurities from the heterogeneous mixture does not includecentrifugation, disk-stack centrifugation, depth filtration, staticfiltration, tangential flow filtration, a hydrocyclone or anycombination thereof. Alternatively, the process described herein maycontinuously receive a heterogeneous mixture containing a biologicalproduct via an input line derived from any continuous large impurityremoval input, for example, without intent to be limiting, a continuous,disk-stack centrifuge and depth filtration process or a hydrocycloneprocess.

In embodiments, the process described herein includes purifying abiological product (e.g. a monoclonal antibody) that is produced in abioreactor. In some embodiments, the process described herein includespurifying a biological product that is continuously produced in abioreactor. For example, the bioreactor includes a bioreactor feed lineand an output bleed line to enable steady-state cell culture growthconditions, and the output bleed line functions as the input line topermit continuous fluid flow from the bioreactor to the dynamicfiltration module. In examples, the bioreactor type includes, but is notlimited to, a fed-batch bioreactor, a perfusion bioreactor, a chemostatbioreactor, or a multi-compartment bioreactor. For example, the flowfrom the bioreactor bleed line is always feeding the downstreampurification system. Alternatively, the process described hereinincludes purifying a biological product (e.g. mRNA) that is not producedin a bioreactor.

In embodiments, provided herein is a process for purifying a biologicalproduct, the method including receiving, via an input line, aheterogeneous mixture containing the biological product, removingimpurities from the heterogeneous mixture by dynamic filtration in adynamic filtration module by feeding the biological product from atleast one output head in fluid communication with the input line to thedynamic filtration module under negative pressure, thereby producing afiltrate comprising the biological product; transferring the filtrate toa first module capable of separating the solution into two or morefractions comprising at least one fraction containing the biologicalproduct, wherein the first module comprises an affinity-based, magneticpurification apparatus, and wherein the first module has at least onefirst inlet and at least one first outlet and is configured to permitfluid flow between the first inlet and the first outlet via a loopconveyor system or a pick and place robotics system; transferring thefraction containing the biological product from the at least one firstoutlet of the first module to a second module having at least one inletfor receiving flow from the at least one first outlet of the firstmodule, wherein the second module comprises a charge-based, magneticpurification apparatus, and wherein the second module has at least onesecond inlet and the at least one second outlet and is configured topermit continuous fluid flow between the second inlet and the secondoutlet via a loop conveyor system or a pick and place robotics system;and thereby purifying the biological product.

In other embodiments, a process for purifying a biological product isprovided. The method includes, receiving, via an input line, aheterogeneous mixture containing the biological product; removingimpurities from the heterogeneous mixture by dynamic filtration in adynamic filtration module by feeding the biological product from atleast one output head in fluid communication with the input line to thedynamic filtration module under negative pressure, thereby producing afiltrate comprising the biological product; transferring the filtrate toa first module capable of separating the solution into two or morefractions comprising at least one fraction containing the biologicalproduct, wherein the first module comprises an affinity-based, magneticpurification apparatus, and wherein the first module has at least onefirst inlet and at least one first outlet and is configured to permitfluid flow between the first inlet and the first outlet via a loopconveyor system or a pick and place robotics system; transferring thefraction containing the biological product from the at least one firstoutlet of the first module to a second module having at least one inletfor receiving flow from the at least one first outlet of the firstmodule, wherein the second module comprises an isoelectric point-basedfluidic purification apparatus, also referred to herein as a free-flowelectrophoresis apparatus, and wherein the second module has at leastone second inlet and at least one second outlet and is configured topermit continuous fluid flow between the second inlet and the secondoutlet; and thereby purifying the biological product.

In embodiments, a process for purifying a biological product isincluded, the method including receiving, via an input line, aheterogeneous mixture containing the biological product; removingimpurities from the heterogeneous mixture by dynamic filtration in adynamic filtration module by feeding the biological product from atleast one output head in fluid communication with the input line to thedynamic filtration module under negative pressure, thereby producing afiltrate comprising the biological product; transferring the filtrate toa first module capable of separating the solution into two or morefractions comprising at least one fraction containing the biologicalproduct, wherein the first module comprises an affinity-basedpurification apparatus, and wherein the first module has at least onefirst inlet and at least one first outlet and is configured to permitfluid flow between the first inlet and the first outlet via a mechanicalrotary system; transferring the fraction containing the biologicalproduct from the at least one first outlet of the first module to asecond module having at least one inlet for receiving flow from the atleast one first outlet of the first module, wherein the second modulecomprises a charge-based purification apparatus, and wherein the secondmodule has at least one second inlet and at least one second outlet andis configured to permit fluid flow between the second inlet and thesecond outlet via a mechanical rotary system; and thereby purifying thebiological product.

In other embodiments, a process for purifying a biological product, isprovided, the method including receiving, via an input line, aheterogeneous mixture containing the biological product; removingimpurities from the heterogeneous mixture by dynamic filtration in adynamic filtration module by feeding the biological product from atleast one output head in fluid communication with the input line to thedynamic filtration module under negative pressure, thereby producing afiltrate comprising the biological product; transferring the filtrate toa first module capable of separating the solution into two or morefractions comprising at least one fraction containing the biologicalproduct, wherein the first module comprises an affinity-basedpurification apparatus, and wherein the first module has at least onefirst inlet and at least one first outlet and is configured to permitfluid flow between the first inlet and the first outlet via a mechanicalrotary system; transferring the fraction containing the biologicalproduct from the at least one first outlet of the first module to asecond module having at least one inlet for receiving flow from the atleast one first outlet of the first module, wherein the second modulecomprises an isoelectric point-based fluidic purification apparatus,also referred to herein as a free-flow electrophoresis apparatus, andwherein the second module has at least one second inlet and at least onesecond outlet and is configured to permit continuous fluid flow betweenthe second inlet and the second outlet; and thereby purifying thebiological product.

In embodiments, a process for purifying a biological product isincluded, the method including receiving, via an input line, aheterogeneous mixture containing the biological product; removingimpurities from the heterogeneous mixture by dynamic filtration in adynamic filtration module by feeding the biological product from atleast one output head in fluid communication with the input line to thedynamic filtration module under negative pressure, thereby producing afiltrate comprising the biological product; transferring the filtrate toa first module capable of separating the solution into two or morefractions comprising at least one fraction containing the biologicalproduct, wherein the first module comprises an affinity-basedpurification apparatus, and wherein the first module has at least onefirst inlet and at least one first outlet and is configured to permitfluid flow between the first inlet and the first outlet via a stagedlinear system; transferring the fraction containing the biologicalproduct from the at least one first outlet of the first module to asecond module having at least one inlet for receiving flow from the atleast one first outlet of the first module, wherein the second modulecomprises a charge-based purification apparatus, and wherein the secondmodule has at least one second inlet and at least one second outlet andis configured to permit fluid flow between the second inlet and thesecond outlet via a staged linear system; and thereby purifying thebiological product.

In other embodiments, a process for purifying a biological product, isprovided, the method including receiving, via an input line, aheterogeneous mixture containing the biological product; removingimpurities from the heterogeneous mixture by dynamic filtration in adynamic filtration module by feeding the biological product from atleast one output head in fluid communication with the input line to thedynamic filtration module under negative pressure, thereby producing afiltrate comprising the biological product; transferring the filtrate toa first module capable of separating the solution into two or morefractions comprising at least one fraction containing the biologicalproduct, wherein the first module comprises an affinity-basedpurification apparatus, and wherein the first module has at least onefirst inlet and at least one first outlet and is configured to permitfluid flow between the first inlet and the first outlet via a stagedlinear system; transferring the fraction containing the biologicalproduct from the at least one first outlet of the first module to asecond module having at least one inlet for receiving flow from the atleast one first outlet of the first module, wherein the second modulecomprises an isoelectric point-based fluidic purification apparatus,also referred to herein as a free-flow electrophoresis apparatus, andwherein the second module has at least one second inlet and at least onesecond outlet and is configured to permit continuous fluid flow betweenthe second inlet and the second outlet; and thereby purifying thebiological product.

In embodiments, a process for purifying a biological product isincluded, the method including receiving, via an input line, aheterogeneous mixture containing the biological product; removingimpurities from the heterogeneous mixture by dynamic filtration in adynamic filtration module by feeding the biological product from atleast one output head in fluid communication with the input line to thedynamic filtration module under negative pressure, thereby producing afiltrate comprising the biological product; transferring the filtrate toa first module capable of separating the solution into two or morefractions comprising at least one fraction containing the biologicalproduct, wherein the first module comprises an affinity-based, fluidicpurification apparatus, and wherein the first module has at least onefirst inlet and at least one first outlet and is configured to permitfluid flow between the first inlet and the first outlet; transferringthe fraction containing the biological product from the at least onefirst outlet of the first module to a second module having at least oneinlet for receiving flow from the at least one first outlet of the firstmodule, wherein the second module comprises a charge-based, fluidicpurification apparatus, and wherein the second module has at least onesecond inlet and at least one second outlet and is configured to permitfluid flow between the second inlet and the second outlet; and therebypurifying the biological product.

In other embodiments, a process for purifying a biological product, isprovided, the method including receiving, via an input line, aheterogeneous mixture containing the biological product; removingimpurities from the heterogeneous mixture by dynamic filtration in adynamic filtration module by feeding the biological product from atleast one output head in fluid communication with the input line to thedynamic filtration module under negative pressure, thereby producing afiltrate comprising the biological product; transferring the filtrate toa first module capable of separating the solution into two or morefractions comprising at least one fraction containing the biologicalproduct, wherein the first module comprises an affinity-based, fluidicpurification apparatus, and wherein the first module has at least onefirst inlet and at least one first outlet and is configured to permitfluid flow between the first inlet and the first outlet; transferringthe fraction containing the biological product from the at least onefirst outlet of the first module to a second module having at least oneinlet for receiving flow from the at least one first outlet of the firstmodule, wherein the second module comprises an isoelectric point-basedfluidic purification apparatus, also referred to herein as a free-flowelectrophoresis apparatus, and wherein the second module has at leastone second inlet and at least one second outlet and is configured topermit continuous fluid flow between the second inlet and the secondoutlet; and thereby purifying the biological product.

In embodiments, a process for purifying a biological product isincluded, the method including receiving, via an input line, aheterogeneous mixture containing the biological product; removingimpurities from the heterogeneous mixture by dynamic filtration in adynamic filtration module by feeding the biological product from atleast one output head in fluid communication with the input line to thedynamic filtration module under negative pressure, thereby producing afiltrate comprising the biological product; transferring the filtrate toa first module capable of separating the solution into two or morefractions comprising at least one fraction containing the biologicalproduct, wherein the first module comprises an affinity-based TFFpurification apparatus, and wherein the first module has at least onefirst inlet and at least one first outlet and is configured to permitfluid flow between the first inlet and the first outlet; transferringthe fraction containing the biological product from the at least onefirst outlet of the first module to a second module having at least oneinlet for receiving flow from the at least one first outlet of the firstmodule, wherein the second module comprises a charge-based TFFpurification apparatus, and wherein the second module has at least onesecond inlet and at least one second outlet and is configured to permitfluid flow between the second inlet and the second outlet; and therebypurifying the biological product.

In other embodiments, a process for purifying a biological product, isprovided, the method including receiving, via an input line, aheterogeneous mixture containing the biological product; removingimpurities from the heterogeneous mixture by dynamic filtration in adynamic filtration module by feeding the biological product from atleast one output head in fluid communication with the input line to thedynamic filtration module under negative pressure, thereby producing afiltrate comprising the biological product; transferring the filtrate toa first module capable of separating the solution into two or morefractions comprising at least one fraction containing the biologicalproduct, wherein the first module comprises an affinity-based TFFpurification apparatus, and wherein the first module has at least onefirst inlet and at least one first outlet and is configured to permitfluid flow between the first inlet and the first outlet; transferringthe fraction containing the biological product from the at least onefirst outlet of the first module to a second module having at least oneinlet for receiving flow from the at least one first outlet of the firstmodule, wherein the second module comprises an isoelectric point-basedfluidic purification apparatus, also referred to herein as a free-flowelectrophoresis apparatus, and wherein the second module has at leastone second inlet and at least one second outlet and is configured topermit continuous fluid flow between the second inlet and the secondoutlet; and thereby purifying the biological product.

Advantages of the process and methods described herein include theability to remove large impurities (e.g., cells, cell debris, andaggregates) without membrane fouling or occlusion. For example, it isknown in the art that clarification of cells, cell debris and aggregatesfrom cell culture media with traditional filtration or tangential flowfiltration systems typically leads to fouling or occlusion of the filtermembrane, thus rendering these methodologies unsuitable as a means tocontinuously remove large impurities from a heterogeneous mixturecontaining a biological product over long-term continuous processing. Incontrast, the dynamic filtration apparatus described herein enablescontinuous removal of large impurities from a heterogeneous mixturecontaining a biological product without membrane fouling, as the activetarget region of the filter membrane is constantly being refreshed.

Additionally, because the entire process of producing and purifying thebiological product may be continuous and can maintain a flow rate thatranges from about 0.1 mL/minute to about 50 mL/minute across theentirety of the process, the process equipment and overall processfootprint is able to have a significantly smaller footprint than currentstandard processes, without sacrificing product throughput or yield on akilogram/year basis. For example, the process for producing andpurifying a monoclonal antibody as described herein is operated with afootprint that occupies up to about 30,000 square feet. In contrast,current monoclonal antibody production and downstream processes requireat least 200,000 square feet. In examples, the process of purifying thebiological product has a flow rate that ranges from about 1 mL/minute toabout 10 mL/minute. In some examples, the flow rate of the step ofcontinuously removing large impurities from the heterogeneous mixtureranges from about 0.1 mL/minute to about 50 mL/minute. In otherexamples, the flow rate of the step of continuously removing largeimpurities from the heterogeneous mixture is equivalent to the flow ratefrom the bioreactor bleed line. In other examples, the process providesthat the flow rate of the step of continuously transferring the filtrateto a first module ranges from about 0.1 mL/minute to about 50 mL/minute.In yet other examples, the process provides that the flow rate of thestep of continuously transferring the fraction containing the biologicalproduct from the first outlet to a second module ranges from about 0.1mL/minute to about 50 mL/minute.

An important advantage of the process and methods utilizing magneticresin beads (e.g. magnetic agarose) or traditional resin beads (e.g.agarose) described herein includes that these systems do not requiretraditional stationary phase or packed resin columns (e.g., for standardchromatographies) to be sanitized, recycled and/or regenerated. Forexample, these systems provide for recycling and/or regeneration of theresin beads (e.g., magnetic or non-magnetic resin beads) to create alimitless surface area of the resin beads during operation, and in turnprovides a continuous and cost-effective method.

Put in another way, the modules described herein do not have a fixedbinding or association capacity. In specific examples, the resin beadsused during purification of the biological product, as described herein,are constantly being recycled and regenerated, and therefore able toaccept flow from the previous step, either a dynamic filtration moduleor a purification module, without interruption of the flow from thebioreactor bleed line. Put another way, the modules described in thepresent invention do not have to be left idle in order to be sanitized,regenerated and/or recycled after running, as they are continuouslyundergoing these steps. The method differs from current continuouschromatographic methods, in that current methods have defined columncapacity limitations due to resin packing constraints and thus requirecolumn switching of multiple packed columns to accept continuous inputflow and enable regeneration and/or recycling of the columns that havereached full capacity.

Another advantage of the methods described herein includes that theresin beads are not packed into a stationary phase, rather the resinbeads have mobility. This mobility of the beads increases their surfacearea that is available for binding or association, as substantially moreof the resin bead surface is exposed and free to bind, e.g., morebiological product can bind to the beads. Additionally, the resin beadsin a traditionally packed column (e.g., where the beads lack mobility,have decreased surface area) and are exposed to a high pressuredifferential in order to generate flow through the column. This highpressure differential damages the integrity of the beads, therebydecreasing the column lifetime. The mobile resin beads in the presentlydescribed invention are subjected to substantially lower pressures whichis much gentler on the fragile beads, resulting in longer lifetimes.Additionally, this mobility makes the beads more likely to beregenerated (e.g., fully regenerated) and returned to their initialcondition. This further adds to the cost-effectiveness of the methodsdescribed herein, e.g., as the resin is utilized more efficiently.

As described herein, the resin beads of the claimed methods andapparatuses are mobile throughout the process. Traditionalchromatographic purification methods require column packing, e.g., wherebeads are sufficiently packed together resulting in a stationary phaseand high density. For example, the beads remain separated (circulatingor dispersed) in solution during the process (e.g., they are discretebeads). Moreover, the mobile beads may also mean that the beads do notaggregate together, e.g., at least two or more beads aggregated orgrouped together. Additionally, the mobile beads may also mean that thebeads may form small aggregates that remain dispersed and free to movewithin a solution. Conversely the beads used herein are not packed, butremain mobile, and free to move within a solution.

An important advantage of the process and methods utilizing free-flowelectrophoresis described herein includes that this system represents a“no product loss” process, in that, there is no need for the product tointeract with a resin or other purifying moieties, as the separationoccurs in aqueous solution according to the physicochemical propertiesof the target biological product via interaction with an electric field.Another advantage is observed in the resolving power (e.g., the abilitypurify products having a high degree of physicochemical similarity) ofthis approach, as a higher purity product is achievable when compared totraditional ion-exchange chromatographies. For example, using thefree-flow electrophoresis module and method herein may achieve puritiesof at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater of the biological product. Moreover, the methods and apparatusesdescribed herein can result in an increased purity (of the biologicalproduct), relative to traditional purification and chromatographicmethods. For example, the term “increased” with respect to a levelrefers to any % increase above a control level (e.g., a level of purityresulting from purification using traditional methods). In variousembodiments, the increased level may be at least or about a 1%, 2%, 3%,4%, or 5% increase in purity, at least or about a 10% increase, at leastor about a 15% increase, at least or about a 20% increase, at least orabout a 25% increase, at least or about a 30% increase, at least orabout a 35% increase, at least or about a 40% increase, at least orabout a 45% increase, at least or about a 50% increase, at least orabout a 55% increase, at least or about a 60% increase, at least orabout a 65% increase, at least or about a 70% increase, at least orabout a 75% increase, at least or about a 80% increase, at least orabout a 85% increase, at least or about a 90% increase, at least orabout a 95% increase, relative to traditional purification methods. Inother examples of the disclosure, the purity of the biological productresulting from the methods and apparatuses described herein is increasedby about 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 2.1×,2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, or 3.0×, compared to thepurity of a biological product using standard commercial orchromatographic techniques.

Additionally, the separation based on intrinsic physicochemicalproperties of the biological product (e.g., isoelectric point, surfacecharge, net charge, zeta potential, electrophoretic mobility,electrostatic interactions, etc. . . . ) extends the utility of thisapproach for the purification a plethora of biological products,including, but not limited to, a protein or fragment thereof (apolypeptide), an antibody or fragment thereof, a cytokine, a chemokine,a growth factor, an enzyme, an oligonucleotide, a virus, an adenovirus,an adeno-associated virus (AAV), or a lentivirus.

Further, the modular approach affords flexibility in process design toaccommodate a diverse range of biological products.

In embodiments, in the process described herein, during the purificationby dynamic filtration, filtrate comprising the biological product iscreated and fed under negative pressure into a vacuum collection vesselcapable of collecting from about 50 mL to about 100 L. In examples, thevacuum collection vessel capable of collecting the filtrate is fromabout 1 L to about 10 L. In other examples, the vacuum collection vesselcapable of collecting the filtrate is from about 1 L to about 50 L.

In embodiments, the dynamic filtration module includes at least oneoutput head for modulating flow of the heterogeneous mixture anddispensing the heterogeneous mixture onto the active target region ofthe filter membrane. In examples, the at least one output head is a tubeor a slot die.

In embodiments, the at least one dynamic filtration module may furtherinclude at least one additional input line to supply a wash buffer via acoaxial output head, a separate monoaxial output head, a separate slotdie output head, a slot die output head with multiple openings, or anycombination thereof.

In some embodiments, the dynamic filtration module includes elementsknown to those skilled in the art, for example, without intent to belimiting, active or passive edge guides, tension control (e.g. adancer), break and tension detectors, or any combination thereof.

In embodiments, the process herein includes that the at least one outputhead (in fluid communication with the input line to the dynamicfiltration module) is capable of xy rastering or rθ rastering. Inexamples, the at least one output head is capable of xy rastering. Insome examples, the at least one output head is capable of rθ rastering.In other examples, the at least one output head is capable of motionalong the z-axis. In yet other examples, the at least one output head iscapable of xy rastering and motion along the z-axis.

In embodiments, the dynamic filtration module includes a filter membraneroll, a membrane support structure, at least one support rod or roller,at least one vacuum line, a vacuum system, and at least one vacuumcollection vessel.

In embodiments, the filter membrane roll includes a rolled filtermembrane, wherein the filter membrane includes, but is not limited to,polyethersulfone (PES), hydrophilic polysulfone, cellulose ester,cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF,polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE,or any combination thereof.

In embodiments, the pore size of the rolled filter membrane depends onthe biological product being purified. In examples, the rolled filtermembrane has a pore size in the range from 0.1 μm to 1 μm.Alternatively, the pore size is in the range from about 0.2 μm to about0.45 μm, or the pore size is less than about 0.45 μm. In other examples,when purifying an antibody, the pore size of the rolled filter membraneis in the range of 0.2 μm to about 0.45 μm.

In embodiments, the filter membrane roll has a width from about 10 mm toabout 600 mm. The width of the filter membrane roll, for example, maydepend on the size of the dynamic filtration system, the size of the atleast one output head, or the membrane support structure.

In embodiments, the filter membrane roll further functions as a feedreel that communicates with a collection reel, meaning the filtermembrane originates from the pre-fabricated roll and spans to aninitially empty collecting roll, thus creating a reel-to-reel system.

In embodiments, the membrane support structure of the dynamic filtrationmodule includes a mechanically smooth contact surface derived from amaterial having a low static coefficient of friction (e.g.polytetrafluoroethylene (PTFE)) and an opening that has continuity withthe vacuum line. For example, the static coefficient of friction rangesfrom about 0.01 to about 0.1, or from about 0.01 to about 0.05, or fromabout 0.05 to about 0.1. In examples, the membrane support structure ofthe dynamic filtration module includes an opening. The opening, forexample, may include a mesh, at least one slot, at least one hole, afrit, a porous material, or any combination thereof.

In embodiments, the membrane support structure of the dynamic filtrationmodule includes a temperature control mechanism. The temperature controlmechanism maintains a temperature from 4° C. to 37° C. For example,during purification of an antibody, the temperature control mechanismmaintains a temperature from 15° C. to 37° C. Exemplary temperaturecontrol mechanisms include, but are not limited to, single loopcontrollers, multi-loop controllers, closed loop controllers,proportional-integral-derivative (PID) controllers, Peltier devices,resistive heating elements, and/or thermal chucks with circulatingwater/propylene glycol jackets.

In embodiments, the at least one support rod or roller of the dynamicfiltration module has a mechanically smooth contact surface derived froma material having a low static coefficient of friction (e.g. PTFE,perfluoroalkoxy alkane (PFA)). For example, the static coefficient offriction ranges from about 0.01 to about 0.1, or from about 0.01 toabout 0.05, or from about 0.05 to about 0.1. In examples, the supportrod or roller may be stationary or may rotate. In some examples, thesupport rod may further include a bearing, for example, a sleevebearing.

In embodiments, the vacuum system of the dynamic filtration modulemaintains a gauge pressure of about −0.05 bar to about −0.98 bar.

In embodiments, the process of continuously removing large impurities(e.g., cells, cell debris, and aggregates) from the heterogeneousmixture by dynamic filtration comprises a multiple stage filtration withat least two discrete rolled filter membranes with different pore sizes.In examples, this multiple stage dynamic filtration process includes atleast one first dynamic filtration apparatus having a rolled filtermembrane with a large pore size (e.g., 0.45 μm) in fluid communicationwith at least one second dynamic filtration apparatus having a rolledfilter membrane with a small pore size (e.g., 0.2 μm), thereby producinga filtrate comprising the biological product. Alternatively, a similarresult could be achieved by a single dynamic filtration apparatus havingat least two rolled filter membranes being fed by separate feed reels,resulting in a layered set of filter membranes across the target region(e.g., active target region), wherein the heterogeneous mixture contactsa larger pore size filter membrane first (e.g., 0.45 μm), followed bycontact with a smaller pore size filter membrane next (e.g., 0.2 μm).

In embodiments, the process described herein includes continuouslytransferring the filtrate to a first module capable of separating thesolution into two or more fractions comprising at least one fractioncontaining the biological product, and the first module includes anaffinity-based, magnetic purification apparatus. For example, theaffinity-based, magnetic purification apparatus further includes asuspension of magnetic resin beads. The surface of the magnetic resinbeads, for example, without intent to be limiting, is coupled withProtein A, Protein G, Protein L, an antigenic protein, a protein, areceptor, an antibody, or an aptamer. In examples, the magnetic resinbeads may be paramagnetic or superparamagnetic.

In examples, the magnetic resin beads of the affinity-based, magneticpurification apparatus have a diameter of about 0.2 micron to about 200micron. In other examples, the beads have a diameter from about 0.2 μmto about 100μM, or from about 1 μm to about 200 μm, or from about 10 μmto about 200 μm, or from about 20 μm to about 200 μm, or from about 30μm to about 200 μm, or from about 50 to about 200 μm, or from about 150to about 200 μm. Alternatively, the beads have a diameter from about 1μm to about 100 μm, or from about 50 μm to about 100 μm. The diameter ofthe magnetic resin beads may depend on the biological product beingpurified and the overall flow rate of the process. For example,purification of a monoclonal antibody may include magnetic resin beadsthat are about 40 to about 90 microns in size. Moreover, the magneticresin beads may have a concentration ranging from about 0.01% to about25% by weight. For example, the concentration of the magnetic resinbeads may be about 1% by weight. In some examples, purification of amonoclonal antibody may include magnetic resin beads that have aconcentration of about 1% to about 10% by weight. In other examples, thebinding capacity of the magnetic resin beads is a function of the beadconcentration, surface area-to-volume ratio, affinity ligand density, orany combination thereof. In yet other examples, the magnetic resin beadsmay be solid, porous, nanoporous, microporous, or any combinationthereof.

In embodiments, the process described herein includes continuouslytransferring the fraction containing the biological product from the atleast one first outlet of the first module to a second module, and wherethe second module includes a charge-based, magnetic purificationapparatus (e.g., a positive and/or negative charge-based, magneticpurification apparatus), the charge-based, magnetic purificationapparatus further comprising magnetic resin beads. For example, thesurface of the magnetic resin beads may have cationic functionality,derived from the coupling of positively charged functional groups, toenable purification based on charge or electrostatic interactions. Forexample, the positively charged functional groups include amines,cationic polymers, net positively charged peptides, net positivelycharged proteins, or any combination thereof. Alternatively, the surfaceof the magnetic resin beads may have anionic functionality, derived fromthe coupling of negatively charged functional groups, to enablepurification based on charge or electrostatic interactions. For example,the negatively charged functional groups include carboxyls, anionicpolymers, net negatively charged peptides, net negatively chargedproteins, oligonucleotides, or any combination thereof. In examples, themagnetic resin beads may be paramagnetic or superparamagnetic.

In embodiments, the magnetic resin beads of the charged-based, magneticpurification apparatus have a diameter of about 0.2 micron to about 200micron. The diameter of the magnetic resin beads may depend on thebiological product being purified and the overall flow rate of theprocess. For example, purification of a monoclonal antibody may includemagnetic resin beads that are about 40 to about 90 microns in size.Moreover, the magnetic resin beads may have a concentration ranging fromabout 0.01% to about 25% by weight. For example, the concentration ofthe magnetic resin beads may be about 1% by weight. In some examples,purification of a monoclonal antibody may include magnetic resin beadsthat have a concentration of about 1% to about 10% by weight. In otherexamples, the charge or electrostatic association capacity of themagnetic resin beads is a function of the bead concentration, surfacearea-to-volume ratio, surface charge density, net charge, or anycombination thereof. In yet other examples, the magnetic resin beads maybe solid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, as described herein, one or both of the first(affinity-based, magnetic purification) and/or second (charged-based,magnetic purification, including a positive and/or negativecharged-based, magnetic purification apparatus) module(s) may alsoinclude at least one external magnetic field. For example, the at leastone external magnetic field includes a permanent magnet or anelectromagnet. The at least one external magnetic field includes amagnetic field strength of about 0.01 Tesla to about 1 Tesla (e.g., upto 1 Tesla). Alternatively, the at least one external magnetic field isshielded.

In embodiments, the loop conveyor system has at least two transportvessels charged with magnetic resin beads that are configured tocontinuously receive a mixture containing a biological product andsubsequently transport the resulting heterogeneous mixture containing abiological product, magnetic resin beads, a buffer, or any combinationthereof. For example, at least one of the at least two transport vesselsis positioned in or within close proximity of an external magnetic fieldto attract the magnetic resin beads.

In embodiments, the pick and place robotics system has at least twotransport vessels charged with magnetic resin beads that are configuredto continuously receive a mixture containing a biological product andsubsequently transport the resulting heterogeneous mixture containing abiological product, magnetic resin beads, a buffer, or any combinationthereof. For example, at least one of the at least two transport vesselsis placed in or within close proximity of an external magnetic field toattract the magnetic resin beads.

In embodiments, the first (affinity-based, magnetic purification) and/orthe second (charge-based, magnetic purification, including a positiveand/or negative charged-based, magnetic purification apparatus) modulefurther includes at least one tangential flow filtration system operatedin fed-batch or perfusion mode. In examples, the tangential flowfiltration system may be used to concentrate and buffer exchange thefraction containing the biological product.

In embodiments, the process described herein includes continuouslytransferring the filtrate to a first module capable of separating thesolution into two or more fractions comprising at least one fractioncontaining the biological product, and the first module includes anaffinity-based purification apparatus. For example, the affinity-basedpurification apparatus further includes a suspension of resin beads. Thesurface of the resin beads, for example, without intent to be limiting,is coupled with Protein A, Protein G, Protein L, an antigenic protein, aprotein, a receptor, an antibody, or an aptamer.

In examples, the resin beads of the affinity-based purificationapparatus have a diameter of about 0.2 micron to about 200 micron. Thediameter of the resin beads may depend on the biological product beingpurified and the overall flow rate of the process. For example,purification of a monoclonal antibody may include resin beads that areabout 90 microns in size. Moreover, the resin beads may have aconcentration ranging from about 0.01% to about 25% by weight. Forexample, the concentration of the resin beads may be about 1% by weight.In some examples, purification of a monoclonal antibody may includeresin beads that have a concentration of about 1% to about 10% byweight. In other examples, the binding capacity of the resin beads is afunction of the bead concentration, surface area-to-volume ratio,affinity ligand density, or any combination thereof.

In yet other examples, the resin beads may be solid, porous, nanoporous,microporous, or any combination thereof.

In embodiments, the process described herein includes continuouslytransferring the fraction containing the biological product from the atleast one first outlet of the first module to a second module, and wherethe second module includes a charge-based purification apparatus (e.g.,a positive and/or negative charged-based purification apparatus), thecharge-based purification apparatus further comprising resin beads. Forexample, the surface of the resin beads may have cationic functionality,derived from the coupling of positively charged functional groups, toenable purification based on charge or electrostatic interactions. Forexample, the positively charged functional groups include amines,cationic polymers, net positively charged peptides, net positivelycharged proteins, or any combination thereof. Alternatively, the surfaceof the resin beads may have anionic functionality, derived from thecoupling of negatively charged functional groups, to enable purificationbased on charge or electrostatic interactions. For example, thenegatively charged functional groups include, carboxyls, anionicpolymers, net negatively charged peptides, net negatively chargedproteins, oligonucleotides, or any combination thereof.

In embodiments, the resin beads of the charged-based purificationapparatus have a diameter of about 0.2 micron to about 200 micron. Thediameter of the resin beads may depend on the biological product beingpurified and the overall flow rate of the process. For example,purification of a monoclonal antibody may include resin beads that areabout 90 microns in size. Moreover, the resin beads may have aconcentration ranging from about 0.01% to about 25% by weight. Forexample, the concentration of the resin beads may be about 1% by weight.In some examples, purification of a monoclonal antibody may includeresin beads that have a concentration of about 1% to about 10% byweight. In other examples, the charge or electrostatic associationcapacity of the resin beads is a function of the bead concentration,surface area-to-volume ratio, surface charge density, net charge, or anycombination thereof. In yet other examples, the resin beads may besolid, porous, nanoporous, microporous, or any combination thereof.

In embodiments, the mechanical rotary system (e.g., which permitscontinuous fluid flow between a first and/or second inlet and the firstand/or second outlet) has at least two vessels including mobile resinbeads (e.g., charged with mobile resin beads) that are configured toreceive a mixture (e.g., continuously receive) containing a biologicalproduct and subsequently transport the resulting heterogeneous mixturecontaining a biological product, resin beads, a buffer, or anycombination thereof, to a designated purification position.

In other embodiments, the system (e.g., a staged linear system whichpermits continuous fluid flow between a first and/or second inlet andthe first and/or second outlet) has at least two vessels with mobileresin beads (e.g., charged with mobile resin beads) that are configuredto receive a mixture (e.g., continuously receive) containing abiological product and subsequently process the resulting mixturecontaining a biological product, resin beads, a buffer, or anycombination thereof.

In embodiments, the first (affinity-based purification) and/or thesecond (charge-based purification, including a positive and/or negativecharged-based purification apparatus) module further includes at leastone tangential flow filtration system operated in fed-batch or perfusionmode to concentrate and buffer exchange the fraction containing thebiological product.

In embodiments, the process described herein includes continuouslytransferring the filtrate to a first module capable of separating thesolution into two or more fractions including at least one fractioncontaining the biological product, wherein the first module is anaffinity-based, fluidic purification apparatus having at least onehybrid fluidic device or chip. In embodiments, the at least one hybridfluidic device or chip has a cross-flow channel, at least one magneticfield, and at least one mechanical force generator. Moreover, the atleast one mechanical force generator can include an ultrasonictransducer or a piezoelectric component capable of generating a defined,unidirectional force. In other examples, the at least one externalmagnetic field comprises a permanent magnet, an electromagnet, apatterned magnet, or combinations thereof. For example, the at least oneexternal magnetic field may have a magnetic field strength of about 0.01Tesla (T) to about 1 Tesla (e.g., up to 1 Tesla). In other examples, themagnetic field strength is about 0.01 T, about 0.1 T, or about 1 T. Inother embodiments, the at least one hybrid fluidic device or chip has across-flow channel, at least one magnetic field, and at least onedielectrophoretic electrode. The at least one dielectrophoreticelectrode is capable of inducing a defined, unidirectional force.Moreover, the at least one external magnetic field comprises a permanentmagnet, an electromagnet, a patterned magnet, or combinations thereof.For example, the at least one external magnetic field may have amagnetic field strength of about 0.01 Tesla about 1 Tesla (e.g., up toabout 1 Tesla).

In embodiments, the affinity-based, fluidic purification apparatus thatfurther comprises magnetic resin beads. The surface of the magneticresin bead for example, without intent to be limiting, is coupled withProtein A, Protein G, Protein L, an antigenic protein, a protein, areceptor, an antibody, or an aptamer. In examples, the magnetic resinbeads may be paramagnetic or superparamagnetic.

In embodiments, the magnetic resin beads of affinity-based, fluidicpurification apparatus have a diameter of about 0.2 micron to about 200micron. For example, purification of a monoclonal antibody may includemagnetic resin beads that are about 40 microns in size. Moreover, themagnetic resin beads may have a concentration ranging from about 0.01%to about 25% by weight. For example, the initial concentration of themagnetic resin beads may be about 1% by weight. In some examples,purification of a monoclonal antibody may include magnetic resin beadsthat have a concentration of about 1% to about 10% by weight. In otherexamples, the binding capacity of the magnetic resin beads is a functionof the bead concentration, surface area-to-volume ratio, affinity liganddensity, or any combination thereof. In yet other examples, the magneticresin beads may be solid, porous, nanoporous, microporous, or anycombination thereof.

In embodiments, the process described herein includes continuouslytransferring the fraction containing the biological product from the atleast one first outlet of the first module to a second module, whereinthe second module includes a charge-based, fluidic purificationapparatus. For example, the charge-based, fluidic purification apparatushas at least one hybrid fluidic device or chip. The at least one hybridfluidic device or chip may have a cross-flow channel, at least onemagnetic field, and at least one mechanical force generator. Moreover,the at least one mechanical force generator comprises an ultrasonictransducer or a piezoelectric component capable of generating a defined,unidirectional force. In other examples, the at least one externalmagnetic field comprises a permanent magnet, an electromagnet, apatterned magnet, or combinations thereof. For example, the at least oneexternal magnetic field may have a magnetic field strength of about 0.01Tesla (T) to about 1 Tesla (e.g., up to about 1 Tesla). In otherexamples, the magnetic field strength is about 0.01 T, about 0.1 T, orabout 1 T. In other embodiments, the hybrid fluidic device or chip mayhave a cross-flow channel, at least one magnetic field, and at least onedielectrophoretic electrode, wherein the at least one dielectrophoreticelectrode is capable of inducing a defined, unidirectional force.Moreover, the at least one external magnetic field comprises a permanentmagnet or an electromagnet. In examples, the at least one externalmagnetic field comprises a magnetic field strength from about 0.01 Teslato about 1 Tesla (e.g., up to about 1 Tesla).

In embodiments, the charge-based, fluidic purification apparatus (e.g.,a positive and/or negative charged-based, fluidic purificationapparatus) further includes a suspension of magnetic resin beads. Thesurface of the magnetic resin beads have cationic functionality, derivedfrom the coupling of positively charged functional groups, to enablepurification based on charge or electrostatic interactions. Thepositively charged functional groups include amines, cationic polymers,net positively charged peptides, net positively charged proteins, or anycombination thereof. Alternatively, the magnetic resin bead surface mayinclude anionic functionality, derived from the coupling of negativelycharged functional groups, to enable purification based on charge orelectrostatic interactions. The negatively charged functional groupsinclude carboxyls, anionic polymers, net negatively charged peptides,net negatively charged proteins, oligonucleotides, or any combinationthereof. In examples, the magnetic resin beads may be paramagnetic orsuperparamagnetic.

In examples, the magnetic resin beads of the charge-based, fluidicpurification apparatus have a diameter of about 0.2 micron to about 200micron. The diameter of the magnetic resin beads may depend on thebiological product being purified and the flow rate of the process. Forexample, purification of a monoclonal antibody may include magneticresin beads that are about 40 microns in size. Moreover, the magneticresin beads may have a concentration ranging from about 0.01% to about25% by weight. For example, the concentration of the magnetic resinbeads may be about 1% by weight. In some examples, purification of amonoclonal antibody may include magnetic resin beads that have aconcentration of about 1% to about 10% by weight. In other examples, thecharge or electrostatic association capacity of the magnetic resin beadsis a function of the bead concentration, surface area-to-volume ratio,surface charge density, net charge, or any combination thereof. In yetother examples, the magnetic resin beads may be solid, porous,nanoporous, microporous, or any combination thereof.

In embodiments, the first (affinity-based, fluidic purification) modulefurther has at least one equilibration vessel to allow for binding ofthe biological product to the magnetic resin bead surface, and at leastone low pH equilibration vessel to allow for de-binding interactions ofthe biological product from the magnetic resin bead surface.

In embodiments, the second (charge-based, fluidic purification,including a positive and/or negative charged-based, fluidic purificationapparatus) module further has at least one association equilibrationvessel to allow for association, based on charge or electrostaticinteractions, of the biological product with the magnetic resin beadsurface, and at least one dissociation equilibration vessel to allow fordissociation of the biological product from the magnetic resin beadsurface. In examples, multiple dissociation equilibration vessels areutilized with multiple charge-based, fluidic purification apparatuses toachieve a gradient dissociation, for example, a pH gradient or an ionicstrength gradient.

In embodiments, the magnetic resin beads as described herein arerecycled, and re-used. For example, the beads may be re-used at least 2,3, 4, or more times for purifying a biological product. To enablerecycling and reuse of the magnetic resin beads, the at least oneregeneration equilibration vessel may be utilized in combination with atangential flow filtration system to concentrate and buffer exchange themagnetic resin beads to return the magnetic resin beads to their initialcondition.

As described herein, the first (affinity-based, fluidic purification)and/or the second (charge-based, fluidic purification, including apositive and/or negative charged-based, fluidic purification apparatus)module includes a hybrid microfluidic, mesofluidic, millifluidic,macrofluidic device or chip, or any combination thereof, to purify abiological product, for example, a hybrid microfluidic device comprisingat least one magnetic field and at least one of a piezoelectriccomponent or a dielectrophoretic electrode.

In embodiments, the first (affinity-based, fluidic purification) and/orthe second (charge-based, fluidic purification, including a positiveand/or negative charged-based, fluidic purification apparatus) modulefurther includes at least one tangential flow filtration system operatedin fed-batch or perfusion mode to concentrate and buffer exchange thefraction containing the biological product.

In embodiments, the process described herein includes continuouslytransferring the filtrate to a first module capable of separating thesolution into two or more fractions comprising at least one fractioncontaining the biological product, and the first module includes anaffinity-based TFF purification apparatus. For example, theaffinity-based TFF purification apparatus has at least three tangentialflow filtration systems in fluid communication.

In embodiments, the affinity-based TFF purification apparatus furtherincludes a suspension of resin beads. The surface of the resin beads,for example, without intent to be limiting, is coupled with Protein A,Protein G, Protein L, an antigenic protein, a protein, a receptor, anantibody, or an aptamer.

In embodiments, the resin beads of the affinity-based TFF purificationapparatus have a diameter of about 10 micron to about 200 micron. Thediameter of the resin beads may depend on the biological product beingpurified and the overall flow rate of the process. For example,purification of a monoclonal antibody may include resin beads that areabout 90 microns in size. Moreover, the resin beads may have aconcentration ranging from about 0.01% to about 25% by weight. Forexample, the concentration of the resin beads may be about 1% to about20% by weight. In other examples, the binding capacity of the resinbeads is a function of the bead concentration, surface area-to-volumeratio, affinity ligand density, or any combination thereof. In yet otherexamples, the resin beads may be solid, porous, nanoporous, microporous,or any combination thereof.

In embodiments, the process described herein includes continuouslytransferring the fraction containing the biological product from the atleast one first outlet of the first module to a second module, andwherein the second module includes a charge-based TFF purificationapparatus (e.g., a positive and/or negative charged-based TFFpurification apparatus). For example, the charge-based TFF purificationapparatus has at least three tangential flow filtration systems in fluidcommunication.

In embodiments, the charge-based TFF purification apparatus furtherincludes a suspension of resin beads. For example, the surface of theresin beads may have cationic functionality, derived from the couplingof positively charged functional groups, to enable purification based oncharge or electrostatic interactions. For example, the positivelycharged functional groups include amines, cationic polymers, netpositively charged peptides, net positively charged proteins, or anycombination thereof. Alternatively, the surface of the resin bead mayhave anionic functionality, derived from the coupling of negativelycharged functional groups, to enable purification based on charge orelectrostatic interactions. For example, the negatively chargedfunctional groups include, carboxyls, anionic polymers, net negativelycharged peptides, net negatively charged proteins, oligonucleotides, orany combination thereof.

In embodiments, the resin beads of the charged-based TFF purificationapparatus have a diameter of about 0.2 micron to about 200 micron. Thediameter of the resin beads may depend on the biological product beingpurified and the overall flow rate of the process. For example,purification of a monoclonal antibody may include resin beads that areabout 90 microns in size. Moreover, the resin beads may have aconcentration ranging from about 0.01% to about 25% by weight. Forexample, the concentration of the resin beads may be about 1% to about20% by weight. In other examples, the charge or electrostaticassociation capacity of the resin beads is a function of the beadconcentration, surface area-to-volume ratio, surface charge density, netcharge, or any combination thereof. In yet other examples, the resinbeads may be solid, porous, nanoporous, microporous, or any combinationthereof.

In embodiments, the first (affinity-based TFF purification) modulefurther has at least one equilibration vessel to allow for binding ofthe biological product to the resin bead surface, and at least one lowpH equilibration vessel to allow for de-binding interactions of thebiological product from the resin bead surface.

In embodiments, the second (charge-based TFF purification, including apositive and/or negative charged-based TFF purification apparatus)module further has at least one association equilibration vessel toallow for association, based on charge or electrostatic interactions, ofthe biological product with the resin bead surface, and at least onedissociation equilibration vessel to allow for dissociation of thebiological product from the resin bead surface. In examples, multipledissociation equilibration vessels are utilized with multiplecharge-based, fluidic purification apparatuses to achieve a gradientdissociation, for example, a pH gradient or an ionic strength gradient.

In embodiments, the resin beads as described herein are recycled, andre-used. For example, the beads may be re-used at least 2, 3, 4, or moretimes for purifying a biological product. To enable recycling and reuseof the resin beads, the at least one regeneration equilibration vesselmay be utilized in combination with a tangential flow filtration systemto concentrate and buffer exchange the resin beads to return the resinbeads to their initial condition.

In embodiments, the first (affinity-based TFF purification) and/or thesecond (charge-based TFF purification, including a positive and/ornegative charged-based TFF purification apparatus) module furtherincludes at least one tangential flow filtration system to concentrateand buffer exchange the fraction containing the biological product.

In other embodiments, the process described herein includes continuouslytransferring the fraction containing the biological product from the atleast one first outlet of the first module to a second module, whereinthe second module includes an isoelectric point-based, fluidicpurification apparatus, also referred to herein as a free-flowelectrophoresis apparatus. For example, the free-flow electrophoresisapparatus has at least one fluidic device comprising a fluidic channelcreated between two parallel plates, an electric field or electric fieldgradient orthogonal to the fluid flow direction, and a pH gradient tooperate in an isoelectric focusing mode of operation. In other examples,the isoelectric point-based, fluidic purification module includes atleast one first fluidic device comprising a fluidic channel createdbetween two parallel plates, an electric field or electric fieldgradient orthogonal to the fluid flow direction, and a coarse pHgradient across the main separation channel (e.g., a pH range from about2 to about 10); and at least one second fluidic device comprising afluidic channel created between two parallel plates, an electric fieldor electric field gradient orthogonal to the fluid flow direction, and afine pH gradient across the main separation channel (e.g., a pH rangefrom about 5 to about 8). In examples, additional, subsequent fluidicdevices or chips comprising a fluidic channel created between twoparallel plates and an electric field or electric field gradientorthogonal to the fluid flow direction may be used to enable furtherrefining of the pH gradient across the main separation channel (e.g., apH range from about 7.1 to about 7.6). Alternatively, the free-flowelectrophoresis apparatus has at least one fluidic device comprising afluidic channel created between two parallel plates, an electric fieldor electric field gradient orthogonal to the fluid flow direction, andno pH gradient to operate in a zone electrophoresis or charge separatingmode of operation.

In other examples, the isoelectric point-based, fluidic purificationmodule includes at least one first fluidic device comprising a fluidicchannel created between two parallel plates, an electric field orelectric field gradient orthogonal to the fluid flow direction, andconstant basic pH (e.g., a pH of greater than 7); and at least onesecond fluidic device comprising a fluidic channel created between twoparallel plates, an electric field or electric field gradient orthogonalto the fluid flow direction, and a constant acidic pH (e.g., a pH ofless than 7). Furthermore, the free-flow electrophoresis apparatus hasat least one fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and both an acidic pH gradientand a basic pH gradient separated by a spacer solution (e.g. a NaClsolution) to operate in an isotachophoresis mode of operation.

In some embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first fluidic device comprising a fluidicchannel created between two parallel plates and an electric field orelectric field gradient orthogonal to the fluid flow direction, and atleast one second fluidic device comprising a fluidic channel createdbetween two parallel plates and an electric field or electric fieldgradient orthogonal to the fluid flow direction, wherein each deviceconnected in series is capable of operating in an independent mode ofoperation to enable purification. For example, the at least one firstfree-flow electrophoresis apparatus may operate in an isoelectricfocusing mode and the at least one second free-flow electrophoresisapparatus may operate in an isotachophoresis mode are operatedsequentially through connection in series to increase separationresolution.

In other embodiments, without intent to be limiting, the isoelectricpoint-based, fluidic purification module includes at least one firstfluidic device comprising fluidic channel having at least onedielectrophoretic electrode capable of inducing a defined,unidirectional force; at least one second fluidic device comprising afluidic channel created between two parallel plates, an electric fieldor electric field gradient orthogonal to the fluid flow direction, and acoarse pH gradient across the main separation channel (e.g., a pH rangefrom about 2 to about 10); and at least one third fluidic devicecomprising a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and a fine pH gradient across the main separation channel(e.g., a pH range from about 5 to about 8). In examples, additional,subsequent fluidic devices or chips comprising a fluidic channel createdbetween two parallel plates and an electric field or electric fieldgradient orthogonal to the fluid flow direction may be used to enablefurther refining of the pH gradient across the main separation channel(e.g., a pH range from about 7.1 to about 7.6).

In yet other embodiments, the isoelectric point-based fluidicpurification apparatus further comprises an active cooling system (e.g.,a Peltier device, a thermal chuck with a circulating water/propyleneglycol jacket) to enable temperature control and Joule heat dissipation.For example, the active cooling system may control cooling and/or Jouleheat dissipation to enable operation in the range from about 4° C. toabout 50° C., preferably from about 4° C. to about 37° C. For example,when isolating a biological product (e.g., a monoclonal antibody), thetemperature is maintained from about 4° C. to about 37° C.

In further embodiments, the process for purifying a biological productmay also include viral inactivation, viral filtration, tangential flowfiltration (TFF), high performance tangential flow filtration (HP-TFF),ultrafiltration/diafiltration (UF/DF), filter sterilization,fill-finish, lyophilization, or any combination thereof, performedsemi-continuously and downstream of the second module.

In examples, the entire process described herein (the process forpurifying a biological product) is performed at a temperature in therange of about 4° C. to about 50° C., preferably from about 4° C. toabout 37° C. Moreover, the commercial production-scale process forpurifying a biological product is conducted in a system with a footprintthat occupies significantly less square footage than current techniques,without sacrificing product throughput or yield on a kilograms/yearbasis. For example, the process for producing, purifying a monoclonalantibody as described herein is operated with a footprint that occupiesup to about 30,000 square feet. In contrast, current monoclonal antibodyproduction and downstream processes require at least 200,000 squarefeet.

The process described herein is used to purify a biological product, andthe biological product includes, but is not limited to, a protein orfragment thereof (a polypeptide), an antibody or fragment thereof, acytokine, a chemokine, an enzyme, a growth factor, an oligonucleotide, avirus, an adenovirus, an adeno-associated virus, or a lentivirus.

Dynamic Filtration Module

In aspects, provided herein is a dynamic filtration module for removinglarge impurities from a biological product in a heterogeneous mixture.The dynamic filtration module continuously feeds the biological productfrom at least one output head in fluid communication with the input lineto the dynamic filtration module under negative pressure.

In embodiments, the dynamic filtration module includes a filter membraneroll, a membrane support structure, at least one support rod or roller,at least one vacuum line, a vacuum system, and at least one vacuumcollection vessel.

The dynamic filtration module includes a rolled filter membraneextending between a feed reel and a collection reel, the filter membranehaving a target region (e.g., an active target region) that isconfigured to receive the heterogeneous mixture. For example, the filtermembrane of the filter membrane roll is made of a suitable material,including, but not limited to, polyethersulfone (PES), hydrophilicpolysulfone, cellulose ester, cellulose acetate, polyvinylidene fluoride(PVDF), hydrophilic PVDF, polycarbonate, nylon, polytetrafluoroethylene(PTFE), or hydrophilic PTFE.

In embodiments, the pore size of the rolled filter membrane depends onthe biological product being purified. In examples, the rolled filtermembrane has a pore size in the range from 0.1 μm to 1 μm.Alternatively, the pore size is in the range from about 0.2 μm to about0.45 μm, or the pore size is less than about 0.45 μm. In other examples,when purifying an antibody, the pore size of the rolled filter membraneis in the range of 0.2 μm to about 0.45 μm.

In embodiments, the filter membrane roll has a width from about 10 mm toabout 600 mm. The width of the filter membrane roll, for example, maydepend on factors such as the size of the dynamic filtration system andthe size of the membrane support structure.

In embodiments, the filter membrane roll further functions as a feedreel that communicates with a collection reel, thus creating areel-to-reel system. In operation, the heterogeneous mixture is appliedto a fresh, unused target region of the filter membrane, herein alsoreferred to as the “target region” (or “active target region”), whereinthe filter membrane is continuously moved at an appropriate transportvelocity across the membrane support structure as a result of thecollection reel collecting the filter membrane portion that has beenused. In examples, the feed reel motion is governed by a Servo motorcoupled with a gear box to limit rotations per minute (RPM) by a ratioof 200:1 to enable low membrane transport velocities with high torque.The collection reel motion is governed by a Servo motor coupled with agear box to limit RPM by a ratio of 200:1 to enable low membranetransport velocities with high torque. Further, the feed reel motor andthe collection reel motor are controlled by a closed-loop controllerthat operates a feedback mechanism to ensure consistent membranetransport velocity with the constantly changing diameters of the filtermembrane roll on both the feed reel and the collection reel duringoperation.

For example, ensuring consistent membrane transport velocity may beaccomplished using a thickness monitoring system or a rotary encoder. Inexamples, the feed reel and the collection reel operate in the samedirection with equivalent velocities. In other examples, the feed reeland the collection reel operate in the same direction with differentvelocities. Other methods of filter membrane transport from the feedreel to the collection reel can be contemplated by those of skill in theart of the coating and converting industry. In other examples, twodynamic filtration systems are run in parallel. For example, the twoparallel dynamic filtration systems may allow for continuous flowthrough the system during the replacement of the spent filter membraneroll. Furthermore, the two parallel dynamic filtration systems may allowfor equilibration of a full vacuum collection vessel to atmosphericpressure to enable fluid flow to the first purification module withoutinterruption of the process of continuously receiving the heterogeneousmixture from the bioreactor bleed line.

Additionally, the dynamic filtration module includes a membrane supportstructure to support the target region (e.g., an active target region)of the filter membrane as it experiences negative pressure. The membranesupport structure is positioned between the feed reel and the collectionreel, has a mechanically smooth contact surface derived from a materialhaving a low static coefficient of friction (e.g. PTFE), and has anopening that has continuity with the vacuum line. In examples, theopening may include a mesh, at least one slot, at least one hole, afrit, a porous material, or any combination thereof.

In embodiments, the at least one support rod or roller of the dynamicfiltration module has a mechanically smooth contact surface derived froma material having a low static coefficient of friction (e.g. PTFE, PFA).In examples, the dynamic filtration module includes at least one supportrod or roller with a mechanically smooth contact surface to stabilizethe motion of the filter membrane across the membrane support structure.

In embodiments, the membrane support structure of the dynamic filtrationmodule includes a temperature control mechanism to maintain desiredtemperature in the presence of evaporative cooling. The temperaturecontrol mechanism maintains a temperature from about 4° C. to about 37°C. For example, during purification of an antibody, the temperaturecontrol mechanism maintains a temperature in a range from about 15° C.to about 37° C.

In embodiments, the dynamic filtration module includes at least oneoutput head for modulating flow of the heterogeneous mixture anddispensing the heterogeneous mixture onto the target region (e.g., anactive target region) of the filter membrane. In examples, the at leastone output head is a tube or a slot die.

In some embodiments, the dynamic filtration module further includes atleast one additional input line to supply a wash buffer via a coaxialoutput head, a separate monoaxial output head, a separate slot dieoutput head, or a slot die output head with multiple openings.

In some embodiments, the dynamic filtration module includes elementsknown in the coating and converting industry, for example, withoutintent to be limiting, active or passive edge guides, tension control(e.g. a dancer), break and tension detectors, or any combinationthereof.

In embodiments, the dynamic filtration module includes a vacuum systemhaving continuity with the membrane support structure to apply negativepressure across the target region (e.g., an active target region) of thefilter membrane, where the negative pressure allows for target region(e.g., an active target region) f the filter membrane across themembrane support structure and enables collection of the filtratecontaining the biological product. In examples, the vacuum system of thedynamic filtration module maintains a gauge pressure of about −0.05 barto about −0.98 bar for continuous filtration.

In embodiments, the dynamic filtration module further includes at leastone vacuum collection vessel configured to collect the filtrate, and atleast one sensor or detector. In examples, two parallel dynamicfiltration systems are run staggered in time to allow for continuousflow through the system following the complete filling and equilibrationto atmospheric pressure of the first vacuum collection vessel.

In embodiments, the process of continuously removing large impurities(e.g., cells, cell debris, and aggregates) from the heterogeneousmixture by dynamic filtration comprises a multiple stage filtration withat least two discrete rolled filter membranes with different pore sizes.In examples, this multiple stage dynamic filtration process includes atleast one first dynamic filtration apparatus having a rolled filtermembrane with a large pore size (e.g., 0.45 μm) in fluid communicationwith at least one second dynamic filtration apparatus having a rolledfilter membrane with a small pore size (e.g., 0.2 μm), thereby producinga filtrate comprising the biological product. Alternatively, a similarresult could be achieved by a single dynamic filtration apparatus havingat least two rolled filter membranes being fed by separate feed reels,resulting in a layered set of filter membranes across the active targetregion, wherein the heterogeneous mixture contacts a larger pore sizefilter membrane first (e.g., 0.45 μm), followed by contact with asmaller pore size filter membrane next (e.g., 0.2 μm).

Affinity-Based, Magnetic Purification Module

In aspects, provided herein is an affinity-based, magnetic purificationmodule for separating a mixture into two or more fractions, where atleast one fraction contains the biological product. The affinity-based,magnetic purification module includes at least one inlet and at leastone outlet configured to permit continuous fluid flow between the atleast one inlet and the at least one outlet and wherein the flow ratemay be, for example, consistent and constant during steady-stateoperation.

In embodiments, the affinity-based, magnetic purification moduleincludes a suspension of magnetic resin beads, wherein the magneticresin bead surface, without intent to be limiting, is coupled to ProteinA, Protein G, Protein L, an antigenic protein, a protein, a receptor, anantibody, or an aptamer configured to selectively bind said biologicalproduct. In examples, the magnetic resin beads are mobile.

Moreover, the affinity-based, magnetic purification module includes aloop conveyor system including at least two transport vessels chargedwith magnetic resin beads that are configured to continuously receive amixture containing a biological product and subsequently transport theresulting heterogeneous mixture containing a biological product,magnetic resin beads, a buffer, or any combination thereof.

Alternatively, the affinity-based, magnetic purification module includesa pick and place robotics system including at least two transportvessels charged with magnetic resin beads that are configured tocontinuously receive a mixture containing a biological product andsubsequently transport the resulting heterogeneous mixture containing abiological product, magnetic resin beads, a buffer, or any combinationthereof.

In embodiments, the affinity-based, magnetic purification moduleincludes at least one external magnetic field that may be used toattract, and thus separate, said magnetic resin beads from theheterogeneous mixture to enable washing. Further, the at least oneexternal magnetic field may be used to attract, and thus separate, saidmagnetic resin beads from the heterogeneous mixture to enable elution ofsaid biological product. Alternatively, the at least one externalmagnetic field may be used to enable recycling of said magnetic resinbeads. In examples, mixing of the magnetic resin beads may beaccomplished by placing the at least one transport vessel between twoseparate and opposing magnetic fields that toggle between states of onand off.

In embodiments, the affinity-based, magnetic purification moduleincludes at least one binding/wash buffer system.

In embodiments, the affinity-based, magnetic purification moduleincludes at least one elution buffer system.

In embodiments, the affinity-based, magnetic purification moduleincludes at least one magnetic resin bead regeneration buffer system.

In embodiments, the affinity-based, magnetic purification moduleincludes at least one aspirator system to remove waste solution from theat least two transport vessels.

In embodiments, the affinity-based, magnetic purification moduleincludes at least one sensor or detector.

In embodiments, the affinity-based, magnetic purification moduleincludes at least one fluid handling pump.

Positive Charge-Based, Magnetic Purification Module

In aspects, provided herein is a positive charge-based, magneticpurification module for separating a mixture into two or more fractions,where at least one fraction contains a biological product. The positivecharge-based, magnetic purification module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation.

In embodiments, the positive charge-based, magnetic purification moduleincludes a suspension of magnetic resin beads, wherein the magneticresin bead surface comprises cationic functionality configured toselectively associate with said biological product at a specific pH andionic strength. In examples, the magnetic resin beads are mobile.

Moreover, the positive charge-based, magnetic purification moduleincludes a loop conveyor system comprising at least two transportvessels charged with magnetic resin beads that are configured tocontinuously receive a mixture containing a biological product andsubsequently transport the resulting heterogeneous mixture containing abiological product, magnetic resin beads, a buffer, or any combinationthereof.

Alternatively, the positive charge-based, magnetic purification moduleincludes a pick and place robotics system comprising at least twotransport vessels charged with magnetic resin beads that are configuredto continuously receive a mixture containing a biological product andsubsequently transport the resulting heterogeneous mixture containing abiological product, magnetic resin beads, a buffer, or any combinationthereof.

In embodiments, the positive charge-based, magnetic purification moduleincludes at least one external magnetic field that may be used toattract, and thus separate, said magnetic resin beads from theheterogeneous mixture to enable washing. Further, the at least oneexternal magnetic field may be used to attract, and thus separate, saidmagnetic resin beads from the heterogeneous mixture to enabledissociation and purification of said biological product. Alternatively,the at least one external magnetic field may be used to enable recyclingof said magnetic resin beads. In examples, mixing of the magnetic resinbeads may be accomplished by placing the at least one transport vesselbetween two separate and opposing magnetic fields that toggle betweenstates of on and off.

In embodiments, the positive charge-based, magnetic purification moduleincludes at least one association/wash buffer system.

In embodiments, the positive charge-based, magnetic purification moduleincludes at least one dissociation buffer system. In examples, multipledissociation buffers varying pH, ionic strength, or any combinationthereof, are utilized sequentially to create a gradient dissociationeffect.

In embodiments, the positive charge-based, magnetic purification moduleincludes at least one magnetic resin bead regeneration buffer system.

In embodiments, the positive charge-based, magnetic purification moduleincludes at least one aspirator system to remove waste solution from theat least two transport vessels.

In embodiments, the positive charge-based, magnetic purification moduleincludes at least one sensor or detector.

In embodiments, the positive charge-based, magnetic purification moduleincludes and at least one fluid handling pump.

Negative Charge-Based, Magnetic Purification Module

In aspects, provided herein is a negative charge-based, magneticpurification module for separating a mixture into two or more fractions,at least one fraction containing a biological product. The negativecharge-based, magnetic purification module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation.

In embodiments, the negative charge-based, magnetic purification moduleincludes a suspension of magnetic resin beads, wherein the magneticresin bead surface comprises anionic functionality configured toselectively associate with said biological product at a specific pH andionic strength. In examples, the magnetic resin beads are mobile.

Moreover, the negative charge-based, magnetic purification moduleincludes a loop conveyor system comprising at least two transportvessels charged with magnetic resin beads that are configured tocontinuously receive a mixture containing a biological product andsubsequently transport the resulting heterogeneous mixture containing abiological product, magnetic resin beads, a buffer, or any combinationthereof.

Alternatively, the negative charge-based, magnetic purification moduleincludes a pick and place robotics system comprising at least twotransport vessels charged with magnetic resin beads that are configuredto continuously receive a mixture containing a biological product andsubsequently transport the resulting heterogeneous mixture containing abiological product, magnetic resin beads, a buffer, or any combinationthereof.

In embodiments, the negative charge-based, magnetic purification moduleincludes at least one external magnetic field that may be used toattract, and thus separate, said magnetic resin beads from theheterogeneous mixture to enable washing. Further, the at least oneexternal magnetic field may be used to attract, and thus separate, saidmagnetic resin beads from the heterogeneous mixture to enabledissociation and purification of said biological product. Alternatively,the at least one external magnetic field may be used to enable recyclingof said magnetic resin beads. In examples, mixing of the magnetic resinbeads may be accomplished by placing the at least one transport vesselbetween two separate and opposing magnetic fields that toggle betweenstates of on and off.

In embodiments, the negative charge-based, magnetic purification moduleincludes at least one association/wash buffer system.

In embodiments, the negative charge-based, magnetic purification moduleincludes at least one dissociation buffer system. In examples, multipledissociation buffers varying pH, ionic strength, or any combinationthereof, are utilized sequentially to create a gradient dissociationeffect.

In embodiments, the negative charge-based, magnetic purification moduleincludes at least one magnetic resin bead regeneration buffer system.

In embodiments, the negative charge-based, magnetic purification moduleincludes at least one aspirator system to remove waste solution from theat least two transport vessels.

In embodiments, the negative charge-based, magnetic purification moduleincludes at least one sensor or detector.

In embodiments, the negative charge-based, magnetic purification moduleincludes at least one fluid handling pump.

Affinity-Based Purification Module

In aspects, provided herein is an affinity-based purification module forseparating a mixture into two or more fractions, where at least onefraction contains the biological product. The affinity-basedpurification module includes at least one inlet and at least one outletconfigured to permit continuous fluid flow between the at least oneinlet and the at least one outlet and wherein the flow rate may be, forexample, consistent and constant during steady-state operation.

In embodiments, the affinity-based purification module includes asuspension of resin beads, wherein the resin bead surface, withoutintent to be limiting, is coupled to Protein A, Protein G, Protein L, anantigenic protein, a protein, a receptor, an antibody, or an aptamer,which is configured to selectively bind said biological product. Inexamples, the resin beads are mobile.

In embodiments, the affinity-based purification module includes a lidsystem having at least one gasketed lid, where the at least one gasketedlid has at least one inlet to introduce a gas to enable control ofpositive head pressure. Furthermore, the lid system has at least onevent port to enable equilibration to atmospheric pressure, at least oneinlet to introduce a suspension of resin beads, at least one inlet toreceive the filtrate containing a biological product, and/or at leasttwo inlets to introduce a buffer system to disperse the resin beads toenable washing of, elution from, or regeneration of said resin beads. Insome embodiments, the at least one gasketed lid also includes a port toaccept an overhead stirring impeller to enable dispersion of the resinbeads. In examples, the lid system has control of motion along thez-axis.

In embodiments, the affinity-based purification module includes amechanical rotary system, for example, a carousel comprising at leasttwo vessels charged with resin beads that are configured to continuouslyreceive a mixture containing a biological product and subsequentlytransport the resulting heterogeneous mixture containing a biologicalproduct, resin beads, a buffer, or any combination thereof. In examples,the carousel is a rotating structure that holds and transports at leasttwo vessels to different process positions. In some examples, themechanical rotary system is configured to mate with the lid system toenable pressurization and liquid handling. In other examples, themechanical rotary system has control of motion or rotation in thexy-plane.

In embodiments, the at least two vessels of the affinity-basedpurification module each have a supported, basement filter or filtermembrane. In examples, the basement filter (or filter membrane) enablesenable retention of the resin beads during process steps of binding,de-binding, washing, elution, and/or regeneration. In examples, the atleast two vessels may further include a valve to control liquid flow.

In other embodiments, the affinity-based purification module includes astaged linear system, for example, at least two vessels charged withresin beads that are configured to continuously receive a mixturecontaining a biological product and subsequently process the resultingheterogeneous mixture containing a biological product, resin beads, abuffer, or any combination thereof. In examples, the at least twovessels are configured to mate with the lid system to enablepressurization and liquid handling.

In embodiments, the affinity-based purification module includes acollection system that interfaces with at least one of the at least twovessels of the mechanical rotary system to enable collection of waste,the fraction containing the biological product, or any combinationthereof. In examples, the collection system has control of motion alongthe z-axis.

In other embodiments, the affinity-based purification module includes acollection system that interfaces with at least one of the at least twovessels of the staged linear system to enable collection of waste, thefraction containing the biological product, or any combination thereof.In examples, the collection system is connected to the at least one ofthe at least two vessels.

In embodiments, the affinity-based purification module includes at leastone gas. In some embodiments, without intent to be limiting the gascomprises filtered nitrogen or compressed dry air. In examples, the gascreates a pressure head of about 0.1 to about 30 psi.

In embodiments, the affinity-based purification module includes at leastone binding/wash buffer system.

In embodiments, the affinity-based purification module includes at leastone low pH elution buffer system.

In embodiments, the affinity-based purification module includes at leastone resin bead regeneration buffer system.

In embodiments, the affinity-based purification module includes at leastone collection vessel.

In embodiments, the affinity-based purification module includes at leastone sensor or detector.

In embodiments, the affinity-based purification module includes at leastone fluid handling pump.

Positive Charge-Based Purification Module

Also provided herein is a positive charge-based purification module forseparating a mixture into two or more fractions, where at least onefraction contains a biological product. The positive charge-basedpurification module includes at least one inlet and at least one outletconfigured to permit continuous fluid flow between the at least oneinlet and the at least one outlet and wherein the flow rate may be, forexample, consistent and constant during steady-state operation.

In embodiments, the positive charge-based purification module includes asuspension of resin beads, wherein the resin bead surface comprisescationic functionality configured to selectively associate with saidbiological product at a specific pH and ionic strength. In examples, theresin beads are mobile.

In embodiments, the positive charge-based purification module includeslid system having at least one gasketed lid, the at least one gasketedlid comprising at least one inlet to introduce a gas to enable controlof positive head pressure; at least one inlet to introduce a suspensionof resin beads; at least one vent port to enable equilibration toatmospheric pressure; at least one inlet to receive the filtratecontaining a biological product; at least two inlets to introduce abuffer system to disperse the resin beads to enable washing of,dissociation from, or regeneration of said resin beads. In someembodiments, the at least one gasketed lid further comprises a port toaccept an overhead stirring impeller to enable dispersion of the resinbeads. In examples, the lid system has control of motion along thez-axis.

In embodiments, the positive charge-based purification module includes amechanical rotary system, for example, a carousel comprising at leasttwo vessels charged with resin beads that are configured to continuouslyreceive a mixture containing a biological product and subsequentlytransport the resulting heterogeneous mixture containing a biologicalproduct, resin beads, a buffer, or any combination thereof. In examples,the carousel is a rotating structure that holds and transports at leasttwo vessels to different process positions. In some examples, themechanical rotary system is configured to mate with the lid system toenable pressurization. In other examples, the mechanical rotary systemhas control of motion or rotation in the xy-plane.

In embodiments, the at least two vessels of the positive charge-basedpurification module each have a supported, basement filter or filtermembrane. In examples, the basement filter (or filter membrane) enablesenable retention of the resin beads during process steps of association,washing, dissociation, and/or regeneration. In examples, the at leasttwo vessels may further include a valve to control liquid flow.

In other embodiments, the positive charge-based purification moduleincludes a staged linear system, for example, at least two vesselscharged with resin beads that are configured to continuously receive amixture containing a biological product and subsequently process theresulting heterogeneous mixture containing a biological product, resinbeads, a buffer, or any combination thereof. In examples, the at leasttwo vessels are configured to mate with the lid system to enablepressurization and liquid handling.

In embodiments, the positive charge-based purification module includes acollection system that interfaces with at least one of the at least twovessels of the mechanical rotary system to enable collection of waste,the fraction containing the biological product, or any combinationthereof. In examples, the collection system has control of motion alongthe z-axis.

In other embodiments, the positive charge-based purification moduleincludes a collection system that interfaces with at least one of the atleast two vessels of the staged linear system to enable collection ofwaste, the fraction containing the biological product, or anycombination thereof. In examples, the collection system is connected tothe at least one of the at least two vessels.

In embodiments, the affinity-based purification module includes at leastone gas. In some embodiments, without intent to be limiting the gascomprises filtered nitrogen or compressed dry air. In examples, the gascreates a pressure head of about 0.1 to about 30 psi.

In embodiments, the positive charge-based purification module includesat least one association/wash buffer system.

In embodiments, the positive charge-based purification module includesat least one dissociation buffer system. In examples, multipledissociation buffers varying pH, ionic strength, or any combinationthereof, are utilized continuously or sequentially to create a gradientdissociation effect.

In embodiments, the positive charge-based purification module includesat least one resin bead regeneration buffer system.

In embodiments, the positive charge-based purification module includesat least one collection vessel.

In embodiments, the positive charge-based purification module includesat least one sensor or detector.

In embodiments, the positive charge-based purification module includesat least one fluid handling pump.

Negative Charge-Based Purification Module

In aspects, provided herein is a negative charge-based purificationmodule for separating a mixture into two or more fractions, where atleast one fraction contains a biological product. The negativecharge-based purification module includes at least one inlet and atleast one outlet configured to permit continuous fluid flow between theat least one inlet and the at least one outlet and wherein the flow ratemay be, for example, consistent and constant during steady-stateoperation.

In embodiments, the negative charge-based purification module includes asuspension of resin beads, wherein the resin bead surface comprisescationic functionality configured to selectively associate with saidbiological product at a specific pH and ionic strength.

In embodiments, the negative charge-based purification module includeslid system having at least one gasketed lid, the at least one gasketedlid comprising at least one inlet to introduce a gas to enable controlof positive head pressure; at least one vent port to enableequilibration to atmospheric pressure; at least one inlet to introduce asuspension of resin beads; at least one inlet to receive the filtratecontaining a biological product; at least two inlets to introduce abuffer system to disperse the resin beads to enable washing of,dissociation from, or regeneration of said resin beads. In someembodiments, the at least one gasketed lid further comprises a port toaccept an overhead stirring impeller to enable dispersion of the resinbeads. In examples, the lid system has control of motion along thez-axis.

In embodiments, the negative charge-based purification module includes amechanical rotary system, for example, a carousel comprising at leasttwo vessels charged with resin beads that are configured to continuouslyreceive a mixture containing a biological product and subsequentlytransport the resulting heterogeneous mixture containing a biologicalproduct, resin beads, a buffer, or any combination thereof. In examples,the carousel is a rotating structure that holds and transports at leasttwo vessels to different process positions. In some examples, themechanical rotary system is configured to mate with the lid system toenable pressurization. In other examples, the mechanical rotary systemhas control of motion or rotation in the xy-plane.

In embodiments, the at least two vessels of the positive charge-basedpurification module each have a supported, basement filter or filtermembrane. In examples, the basement filter (or filter membrane) enablesenable retention of the resin beads during process steps of association,washing, dissociation, and/or regeneration. In examples, the at leasttwo vessels may further include a valve to control liquid flow.

In other embodiments, the positive charge-based purification moduleincludes a staged linear system, for example, at least two vesselscharged with resin beads that are configured to continuously receive amixture containing a biological product and subsequently process theresulting heterogeneous mixture containing a biological product, resinbeads, a buffer, or any combination thereof. In examples, the at leasttwo vessels are configured to mate with the lid system to enablepressurization and liquid handling.

In embodiments, the positive charge-based purification module includes acollection system that interfaces with at least one of the at least twovessels of the mechanical rotary system to enable collection of waste,the fraction containing the biological product, or any combinationthereof. In examples, the collection system has control of motion alongthe z-axis.

In other embodiments, the positive charge-based purification moduleincludes a collection system that interfaces with at least one of the atleast two vessels of the staged linear system to enable collection ofwaste, the fraction containing the biological product, or anycombination thereof. In examples, the collection system is connected tothe at least one of the at least two vessels.

In embodiments, the affinity-based purification module includes at leastone gas. In some embodiments, without intent to be limiting the gascomprises filtered nitrogen or compressed dry air. In examples, the gascreates a pressure head of about 0.1 to about 30 psi.

In embodiments, the negative charge-based purification module includesat least one association/wash buffer system.

In embodiments, the negative charge-based purification module includesat least one dissociation buffer system. In examples, multipledissociation buffers varying pH, ionic strength, or any combinationthereof, are utilized continuously or sequentially to create a gradientdissociation effect.

In embodiments, the negative charge-based purification module includesat least one resin bead regeneration buffer system.

In embodiments, the negative charge-based purification module includesat least one collection vessel.

In embodiments, the negative charge-based purification module includesat least one sensor or detector.

In embodiments, the negative charge-based purification module includesat least one fluid handling pump.

Affinity-Based, Fluidic Purification Module

In aspects, provided herein is an affinity-based, fluidic purificationmodule for separating a mixture into two or more fractions, at least onefraction containing a biological product. The affinity-based, fluidicpurification module includes at least one inlet and at least one outletconfigured to permit continuous fluid flow between the at least oneinlet and the at least one outlet and wherein the flow rate may be, forexample, consistent and constant during steady-state operation.

In embodiments, the affinity-based, fluidic purification module includesa suspension of magnetic resin beads, wherein the magnetic resin beadsurface, without intent to be limiting, is coupled to Protein A, ProteinG, Protein L, an antigenic protein, a protein, a receptor, an antibody,or an aptamer configured to selectively bind said biological product. Inexamples, the magnetic resin beads are mobile.

In embodiments, the affinity-based, fluidic purification module includesat least one equilibration vessel to allow for binding of the biologicalproduct to the magnetic resin bead surface; and, at least one firsthybrid cross-flow fluidic device comprising a cross-flow channel, atleast one magnetic field, and at least one of a piezoelectric componentor a dielectrophoretic electrode configured to generate or induce aunidirectional force to separate said biological product bound magneticresin beads from said heterogeneous mixture.

In embodiments, the affinity-based, fluidic purification module furtherincludes at least one low pH equilibration vessel to allow forde-binding of the biological product from the magnetic resin beadsurface; and, at least one second hybrid cross-flow fluidic devicecomprising a cross-flow channel, at least one magnetic field, and atleast one of a piezoelectric component or a dielectrophoretic electrodeconfigured to generate or induce a unidirectional force to separate saidmagnetic resin beads from said unbound biological product and completeits elution.

In embodiments, the affinity-based, fluidic purification module furtherincludes at least one tangential flow filtration system operated infed-batch or perfusion mode to concentrate and buffer exchange thefraction containing the biological product.

In embodiments, the affinity-based, fluidic purification module includesat least two buffer systems.

In embodiments, the affinity-based, fluidic purification module includesat least one magnetic resin bead regeneration buffer system.

In embodiments, the affinity-based, fluidic purification module includesat least one equilibration vessel configured to enable recycling of saidmagnetic resin beads.

In embodiments, the affinity-based, fluidic purification module includesat least one sensor or detector.

In embodiments, the affinity-based, fluidic purification module includesat least one fluid handling pump.

Positive Charge-Based, Fluidic Purification Module

In aspects, provided herein is a positive charge-based, fluidicpurification module for separating a mixture into two or more fractions,at least one fraction containing a biological product. The positivecharge-based, fluidic purification module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation.

In embodiments, the positive charge-based, fluidic purification moduleincludes a suspension of magnetic resin beads, wherein the magneticresin bead surface comprises cationic functionality configured toselectively associate with said biological product at a specific pH andionic strength. In examples, the magnetic resin beads are mobile.

In embodiments, the positive charge-based, fluidic purification moduleincludes at least one association equilibration vessel to allow forassociation of the biological product with the magnetic resin beadsurface; and, at least one first hybrid cross-flow fluidic devicecomprising a cross-flow channel, at least one magnetic field, and atleast one of a piezoelectric component or a dielectrophoretic electrodeconfigured to generate or induce a unidirectional force to separate saidbiological product associated magnetic resin beads from saidheterogeneous mixture.

In embodiments, the positive charge-based, fluidic purification moduleincludes at least one dissociation equilibration vessel to allow fordissociation of the biological product from the magnetic resin beadsurface; and, at least one second hybrid cross-flow fluidic devicecomprising a cross-flow channel, at least one magnetic field, and atleast one of a piezoelectric component or a dielectrophoretic electrodeconfigured to generate or induce a unidirectional force to separate saidmagnetic resin beads from said dissociated biological product andcomplete its purification. In examples, multiple dissociationequilibration vessels comprising discrete buffers varying pH, ionicstrength, or any combination thereof, are utilized sequentially tocreate a gradient dissociation effect.

In embodiments, the positive charge-based, fluidic purification modulefurther includes at least one tangential flow filtration system operatedin fed-batch or perfusion mode to concentrate and buffer exchange thefraction containing the biological product.

In embodiments, the positive charge-based, fluidic purification moduleincludes at least two buffer systems.

In embodiments, the positive charge-based, fluidic purification moduleincludes at least one magnetic resin bead regeneration buffer system.

In embodiments, the positive charge-based, fluidic purification moduleincludes at least one equilibration vessel configured to enablerecycling of said magnetic resin beads.

In embodiments, the positive charge-based, fluidic purification moduleincludes at least one sensor or detector.

In embodiments, the positive charge-based, fluidic purification moduleincludes at least one fluid handling pump.

Negative Charge-Based, Fluidic Purification Module

In aspects, provided herein is a negative charge-based, fluidicpurification module for separating a mixture into two or more fractions,at least one fraction containing a biological product. The negativecharge-based, fluidic purification module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation.

In embodiments, the negative charge-based, fluidic purification moduleincludes a suspension of magnetic resin beads, wherein the magneticresin bead surface comprises anionic functionality configured toselectively associate with said biological product at a specific pH andionic strength. In examples, the magnetic resin beads are mobile.

In embodiments, the negative charge-based, fluidic purification moduleincludes at least one association equilibration vessel to allow forassociation of the biological product with the magnetic resin beadsurface; and, at least one first hybrid cross-flow fluidic devicecomprising a cross-flow channel, at least one magnetic field, and atleast one of a piezoelectric component or a dielectrophoretic electrodeconfigured to generate or induce a unidirectional force to separate saidbiological product associated magnetic resin beads from saidheterogeneous mixture.

In embodiments, the negative charge-based, fluidic purification moduleincludes at least one dissociation equilibration vessel to allow fordissociation of the biological product from the magnetic resin beadsurface; and, at least one second hybrid cross-flow fluidic devicecomprising a cross-flow channel, at least one magnetic field, and atleast one of a piezoelectric component or a dielectrophoretic electrodeconfigured to generate or induce a unidirectional force to separate saidmagnetic resin beads from said dissociated biological product andcomplete its purification. In examples, multiple dissociationequilibration vessels comprising discrete buffers varying pH, ionicstrength, or any combination thereof, are utilized sequentially tocreate a gradient dissociation effect.

In embodiments, the negative charge-based, fluidic purification modulefurther includes at least one tangential flow filtration system operatedin fed-batch or perfusion mode to concentrate and buffer exchange thefraction containing the biological product.

In embodiments, the negative charge-based, fluidic purification moduleincludes at least two buffer systems.

In embodiments, the negative charge-based, fluidic purification moduleincludes at least one magnetic resin bead regeneration buffer system.

In embodiments, the negative charge-based, fluidic purification moduleincludes at least one equilibration vessel configured to enablerecycling of said magnetic resin beads.

In embodiments, the negative charge-based, fluidic purification moduleincludes at least one sensor or detector.

In embodiments, the negative charge-based, fluidic purification moduleincludes at least one fluid handling pump.

Affinity-Based TFF Purification Module

In aspects, provided herein is an affinity-based TFF purification modulefor separating a mixture into two or more fractions, at least onefraction containing a biological product. The affinity-based TFFpurification module includes at least one inlet and at least one outletconfigured to permit continuous fluid flow between the at least oneinlet and the at least one outlet and wherein the flow rate isconsistent and constant during steady-state operation.

In embodiments, the affinity-based TFF purification module includes asuspension of resin beads, wherein the resin bead surface, withoutintent to be limiting, is coupled to Protein A, Protein G, Protein L, anantigenic protein, a protein, a receptor, an antibody, or an aptamerconfigured to selectively bind said biological product. In examples, themagnetic resin beads are mobile.

In embodiments, the affinity-based TFF purification module includes atleast one equilibration vessel to allow for binding of the biologicalproduct to the resin bead surface; and, at least one first tangentialflow filtration system to separate said biological product bound resinbeads from said heterogeneous mixture.

In embodiments, the affinity-based TFF purification module furtherincludes at least one low pH equilibration vessel to allow forde-binding of the biological product from the resin bead surface; and,at least one second tangential flow filtration system to separate saidresin beads from said unbound biological product and complete itselution.

In embodiments, the affinity-based TFF purification module includes atleast one regeneration equilibration vessel; and at least one thirdtangential flow filtration system to allow for concentration and bufferexchange of the resin beads to return the resin beads to their initialcondition, thus enabling recycling and reuse of the resin beads.

In embodiments, the affinity-based TFF purification module includes atleast one collection vessel; and at least one fourth tangential flowfiltration system to allow for concentration and buffer exchange of thebiological product, thus purifying the biological product.

In embodiments, the at least one equilibration vessel, the at least onelow pH equilibration vessel, and the at least one regenerationequilibration vessel of the affinity-based TFF purification module maycomprise a single vessel that is transitioned between the correspondingtangential flow filtration systems to enable purification andregeneration of the resin beads with appropriate buffers, whilemaintaining continuous flow of the filtrate via at least one additionalvessel on a parallel flow path.

In embodiments, the regeneration of the resin beads may be accomplishedwith the at least one low pH equilibration vessel and the at least onesecond tangential flow filtration system of the affinity-based TFFpurification module configured to comprise both the low pH elutionbuffer and the regeneration buffer to enable purification, concentrationand buffer exchange, thus regenerating the resin beads withoutnecessitating a separate regeneration equilibration vessel andcorresponding tangential flow filtration system.

In embodiments, the affinity-based TFF purification module includes atleast two buffer systems.

In embodiments, the affinity-based TFF purification module includes atleast one resin bead regeneration buffer system.

In embodiments, the affinity-based TFF purification module includes atleast one hollow fiber membrane filter.

In embodiments, the affinity-based TFF purification module includes atleast one sensor or detector.

In embodiments, the affinity-based TFF purification module includes atleast one fluid handling pump.

Positive Charge-Based TFF Purification Module

In aspects, provided herein is a positive charge-based TFF purificationmodule for separating a mixture into two or more fractions, at least onefraction containing a biological product. The positive charge-based TFFpurification module includes at least one inlet and at least one outletconfigured to permit continuous fluid flow between the at least oneinlet and the at least one outlet and wherein the flow rate isconsistent and constant during steady-state operation.

In embodiments, the positive charge-based TFF purification moduleincludes a suspension of resin beads, wherein the resin bead surfacecomprises cationic functionality configured to selectively associatewith said biological product at a specific pH and ionic strength. Inexamples, the magnetic resin beads are mobile.

In embodiments, the positive charge-based TFF purification moduleincludes at least one association equilibration vessel to allow forassociation of the biological product with the resin bead surface; and,at least one first tangential flow filtration system to separate saidbiological product associated resin beads from said heterogeneousmixture.

In embodiments, the positive charge-based TFF purification moduleincludes at least one dissociation equilibration vessel to allow fordissociation of the biological product from the resin bead surface; and,at least one second tangential flow filtration system to separate saidresin beads from said dissociated biological product and complete itspurification. In some aspects, multiple dissociation equilibrationvessels are utilized with multiple tangential flow filtration systems toachieve a gradient dissociation, for example, a pH gradient or an ionicstrength gradient.

In embodiments, the positive charge-based TFF purification moduleincludes at least one regeneration equilibration vessel; and at leastone third tangential flow filtration system to allow for concentrationand buffer exchange of the resin beads to return the resin beads totheir initial condition, thus enabling recycling and reuse of the resinbeads.

In embodiments, the positive charge-based TFF purification moduleincludes at least one collection vessel; and at least one fourthtangential flow filtration system to allow for concentration and bufferexchange of the biological product, thus purifying the biologicalproduct.

In embodiments, the at least one association equilibration vessel, theat least one dissociation vessel, and the at least one regenerationequilibration vessel of the positive charge-based TFF purificationmodule may comprise a single vessel that is transitioned between thecorresponding tangential flow filtration systems to enable purificationand regeneration of the resin beads with appropriate buffers, whilemaintaining continuous flow of the filtrate via at least one additionalvessel on a parallel flow path.

In embodiments, the regeneration of the resin beads may be accomplishedwith the at least one dissociation vessel and the at least one secondtangential flow filtration system of the positive charge-based TFFpurification module configured to comprise both the dissociation bufferand the regeneration buffer to enable purification, concentration andbuffer exchange, thus regenerating the resin beads without necessitatinga separate regeneration equilibration vessel and correspondingtangential flow filtration system.

In embodiments, the positive charge-based TFF purification moduleincludes at least two buffer systems.

In embodiments, the positive charge-based TFF purification moduleincludes at least one resin bead regeneration buffer system.

In embodiments, the positive charge-based TFF purification moduleincludes at least one hollow fiber membrane filter.

In embodiments, the positive charge-based TFF purification moduleincludes at least one sensor or detector.

In embodiments, the positive charge-based TFF purification moduleincludes at least one fluid handling pump.

Negative Charge-Based TFF Purification Module

In aspects, provided herein is a negative charge-based TFF purificationmodule for separating a mixture into two or more fractions, at least onefraction containing a biological product. The negative charge-based TFFpurification module includes at least one inlet and at least one outletconfigured to permit continuous fluid flow between the at least oneinlet and the at least one outlet and wherein the flow rate isconsistent and constant during steady-state operation.

In embodiments, the negative charge-based TFF purification moduleincludes a suspension of resin beads, wherein the resin bead surfacecomprises anionic functionality configured to selectively associate withsaid biological product at a specific pH and ionic strength.

In embodiments, the negative charge-based TFF purification moduleincludes at least one association equilibration vessel to allow forassociation of the biological product with the resin bead surface; and,at least one first tangential flow filtration system to separate saidbiological product associated resin beads from said heterogeneousmixture.

In embodiments, the negative charge-based TFF purification moduleincludes at least one dissociation equilibration vessel to allow fordissociation of the biological product from the resin bead surface; and,at least one second tangential flow filtration system to separate saidresin beads from said dissociated biological product and complete itspurification. In some aspects, multiple dissociation equilibrationvessels are utilized with multiple tangential flow filtration systems toachieve a gradient dissociation, for example, a pH gradient or an ionicstrength gradient.

In embodiments, the negative charge-based TFF purification moduleincludes at least one regeneration equilibration vessel; and at leastone third tangential flow filtration system to allow for concentrationand buffer exchange of the resin beads to return the resin beads totheir initial condition, thus, enabling recycling and reuse of the resinbeads.

In embodiments, the negative charge-based TFF purification moduleincludes at least one collection vessel; and at least one fourthtangential flow filtration system to allow for concentration and bufferexchange of the biological product, thus purifying the biologicalproduct.

In embodiments, the at least one association equilibration vessel, theat least one dissociation vessel, and the at least one regenerationequilibration vessel of the negative charge-based TFF purificationmodule may comprise a single vessel that is transitioned between thecorresponding tangential flow filtration systems to enable purificationand regeneration of the resin beads with appropriate buffers, whilemaintaining continuous flow of the filtrate via at least one additionalvessel on a parallel flow path.

In embodiments, the regeneration of the resin beads may be accomplishedwith the at least one dissociation vessel and the at least one secondtangential flow filtration system of the negative charge-based TFFpurification module configured to comprise both the dissociation bufferand the regeneration buffer to enable purification, concentration andbuffer exchange, thus regenerating the resin beads without necessitatinga separate regeneration equilibration vessel and correspondingtangential flow filtration system.

In embodiments, the negative charge-based TFF purification moduleincludes at least two buffer systems.

In embodiments, the negative charge-based TFF purification moduleincludes at least one resin bead regeneration buffer system.

In embodiments, the negative charge-based TFF purification moduleincludes at least one hollow fiber membrane filter.

In embodiments, the negative charge-based TFF purification moduleincludes at least one sensor or detector.

In embodiments, the negative charge-based TFF purification moduleincludes at least one fluid handling pump.

Isoelectric Point-Based, Fluidic Purification Module

In aspects, provided herein is an isoelectric point-based, fluidicpurification module for separating a mixture into two or more fractions,at least one fraction containing a biological product. The isoelectricpoint-based fluidic purification module includes at least one inlet andat least one outlet configured to permit continuous fluid flow betweenthe at least one inlet and the at least one outlet and wherein the flowrate may be, for example, consistent and constant during steady-stateoperation.

In embodiments, the process described herein of continuouslytransferring the fraction containing the biological product from the atleast one first outlet of the first module to a second module, whereinthe second module includes free-flow electrophoresis apparatus. Forexample, the free-flow electrophoresis apparatus has at least onefluidic device comprising a fluidic channel created between two parallelplates, an electric field or electric field gradient orthogonal to thefluid flow direction, and an aqueous solution (e.g., an ionic solution,or a solution providing a buffer or ampholyte). In examples, thesolution contacting surfaces of the two parallel plates comprise glass,ceramic, plastic, or any combination thereof. In some examples, theaqueous ionic solution may give rise to a pH gradient. In otherexamples, the aqueous ionic solution may confer constant pH.

In embodiments, the free-flow electrophoresis apparatus has at least onefluidic device comprising a fluidic channel created between two parallelplates, an electric field or electric field gradient orthogonal to thefluid flow direction, and a pH gradient. In examples, the isoelectricpoint-based, fluidic purification module includes at least one firstfluidic device comprising a fluidic channel created between two parallelplates, an electric field or electric field gradient orthogonal to thefluid flow direction, and a coarse pH gradient across the mainseparation channel (in examples, a coarse pH gradient may be a pH rangefrom about 2 to about 10); and at least one second fluidic devicecomprising a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and a fine pH gradient across the main separation channel (inexamples, a fine pH gradient may be a pH range from about 5 to about 8).In examples, additional, subsequent fluidic devices or chips comprisinga fluidic channel created between two parallel plates and an electricfield or electric field gradient orthogonal to the fluid flow directionmay be used to enable further refining of the pH gradient across themain separation channel (e.g., a pH range from about 7.1 to about 7.6).

In other embodiments, the free-flow electrophoresis apparatus has atleast one fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and no pH gradient to operate ina zone electrophoresis or charge separating mode of operation. Inexamples, the isoelectric point-based, fluidic purification moduleincludes at least one first fluidic device comprising a fluidic channelcreated between two parallel plates, an electric field or electric fieldgradient orthogonal to the fluid flow direction, and constant basic pH(e.g., a pH of greater than 7); and at least one second fluidic devicecomprising a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and a constant acidic pH (e.g., a pH of less than 7).

In other embodiments, the free-flow electrophoresis apparatus has atleast one fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and both an acidic pH gradientand a basic pH gradient separated by a spacer solution (e.g. NaClsolution) to operate in an isotachophoresis mode of operation.

In other embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first free-flow electrophoresis apparatuscomprising a fluidic channel created between two parallel plates and anelectric field or electric field gradient orthogonal to the fluid flowdirection, and at least one second free-flow electrophoresis apparatuscomprising a fluidic channel created between two parallel plates and anelectric field or electric field gradient orthogonal to the fluid flowdirection, wherein each device connected in series and is capable ofoperating in an independent mode of operation to enable purification.For example, the at least one first free-flow electrophoresis apparatusmay operate in an isoelectric focusing mode and the at least one secondfree-flow electrophoresis apparatus may operate in an isotachophoresismode to increase separation resolution.

In other embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first fluidic device comprising fluidicchannel having at least one dielectrophoretic electrode capable ofinducing a defined, unidirectional force; at least one second free-flowelectrophoresis apparatus comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and a coarse pH gradient acrossthe main separation channel (e.g., a pH range from about 2 to about 10);and at least one third free-flow electrophoresis apparatus comprising afluidic channel created between two parallel plates, an electric fieldor electric field gradient orthogonal to the fluid flow direction, and afine pH gradient across the main separation channel (e.g., a pH rangefrom about 5 to about 8). In examples, additional, subsequent fluidicdevices or chips comprising a fluidic channel created between twoparallel plates and an electric field or electric field gradientorthogonal to the fluid flow direction may be used to enable furtherrefining of the pH gradient across the main separation channel (e.g., apH range from about 7.1 to about 7.6).

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least two electrodes (e.g. platinum wireelectrodes) to function as an anode or a cathode.

In embodiments, the backpressure within the isoelectric point-basedfluidic purification apparatus is dependent on the channel geometry anddimensions, the inlet and outlet opening and/or tubing diameters, andthe input flow rate. In examples, the backpressure ranges from about 0.5psi to about 10 psi. In some examples, the backpressure is controlledby, for example, without intent to be limiting, a needle valve.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least one de-bubbler system tocontinuously remove O₂ and H₂ gas bubbles that evolve in the electrodechannels under applied voltage. In some embodiments, removal ofelectrolysis bubbles is essential to enable continuous operation forsubstantially long periods of time. In examples, the de-bubbler systemutilizes a hydrophobic PTFE membrane to create a water-tight seal atopthe electrode channel that permits continuous removal of electrolysisbubbles at the point of generation by exposure to a vacuum system. Inexamples, the vacuum gauge pressure ranges from about −0.05 bar to about−0.4 bar.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises an active cooling system or heat sink toenable temperature control and Joule heat dissipation. In examples, theactive cooling system comprises an aluminum thermal chuck containing achilled, circulating water/propylene glycol jacket.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one buffer or ampholyte system.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one electrode solution. In some embodiments, the atleast one electrode solution comprises an electrolyte solutionconfigured to contact and enable the appropriate function of an anode ora cathode, for example, phosphoric acid and sodium hydroxide,respectively. In other embodiments, the at least one electrode solutioncomprises at least one ampholyte solution configured to contact andenable the appropriate function of an anode or a cathode, for example,Tris buffered saline flowing through the main separation channel, theanode channel, and the cathode channel.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one sensor or detector. In examples, the at least onesensor or detector is positioned in-line. In some examples, the at leastone sensor or detector includes, but is not limited to, a flow sensor, atemperature sensor, a conductivity sensor, a pH sensor, a refractiveindex detector, a UV detector, or a backpressure sensor.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one liquid circuit breaker or disconnect downstream ofthe device and upstream of the at least one in-line sensor or detectorto ensure the ability to perform sensing or detection in a voltage-freesolution.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one fluid handling pump.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one collection vessel.

Methods

Provided herein are methods of purifying a biological product from aheterogeneous mixture derived from a bioreactor producing saidbiological product comprising utilizing the processes described herein.In examples, the bioreactor type includes, but is not limited to, abatch bioreactor, a fed-batch bioreactor, a perfusion bioreactor, achemostat bioreactor, or a multi-compartment bioreactor. In someexamples, the bioreactor produce said biological product atsteady-state.

In embodiments, provided herein is a method of purifying a biologicalproduct from a heterogeneous mixture derived from a bioreactor producingsaid biological product comprising utilizing at least one of the modulesdescribed herein, for example, the dynamic filtration module, theaffinity-based, magnetic purification module, the positive charge-based,magnetic purification module, the negative charge-based, magneticpurification module, the affinity-based purification module, thepositive charge-based purification module, the negative charge-basedpurification module, the affinity-based, fluidic purification module,the positive charge-based, fluidic purification module, the negativecharge-based, fluidic purification module, the affinity-based TFFpurification module, the positive charge-based TFF purification module,the negative charge-based TFF purification module, and/or theisoelectric point-based, fluidic purification module.

In some embodiments, provided herein is a method of continuouslypurifying a biological product from a heterogeneous mixture derived froma bioreactor producing said biological product at steady-statecomprising utilizing at least one of the modules described herein, forexample, the dynamic filtration module, the affinity-based, magneticpurification module, the positive charge-based, magnetic purificationmodule, the negative charge-based, magnetic purification module, theaffinity-based purification module, the positive charge-basedpurification module, the negative charge-based purification module, theaffinity-based, fluidic purification module, the positive charge-based,fluidic purification module, the negative charge-based, fluidicpurification module, the affinity-based TFF purification module, thepositive charge-based TFF purification module, the negative charge-basedTFF purification module, and/or the isoelectric point-based, fluidicpurification module.

In other embodiments, provided herein is a method of purifying abiological product from a heterogeneous mixture not derived from abioreactor producing said biological product at steady-state comprisingutilizing at least one of the modules described herein, for example, thedynamic filtration module, the affinity-based, magnetic purificationmodule, the positive charge-based, magnetic purification module, thenegative charge-based, magnetic purification module, the affinity-basedpurification module, the positive charge-based purification module, thenegative charge-based purification module, the affinity-based, fluidicpurification module, the positive charge-based, fluidic purificationmodule, the negative charge-based, fluidic purification module, theaffinity-based TFF purification module, the positive charge-based TFFpurification module, the negative charge-based TFF purification module,and/or the isoelectric point-based, fluidic purification module.

Other aspects of the invention are disclosed infra.

DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D show schematics of exemplary continuous process flowsdescribed herein. FIG. 1A shows an exemplary continuous process flowwherein Step 1 comprises a bioreactor producing a biological product atsteady-state, Step 2 comprises a continuous dynamic filtration module,Step 3 comprises an affinity-based, magnetic purification module, Step 4comprises at least one charge-based, magnetic purification module, Step5 comprises a standard industry viral inactivation and filtrationprocess, for example, performed in fed-batch or perfusion mode, and Step6 comprises high performance tangential flow filtration with a chargedmembrane, for example, performed in fed-batch or perfusion mode toprepare for the standard industry fill-finish process in Step 7. FIG. 1Bshows an exemplary continuous process flow wherein Step 1 comprises abioreactor producing a biological product at steady-state, Step 2comprises a continuous dynamic filtration module, Step 3 comprises anaffinity-based, magnetic purification module, Step 4 comprises apositive charge-based, magnetic purification module, Step 5 comprises anegative charge-based, magnetic purification module, and Step 6comprises high performance tangential flow filtration with a chargedmembrane, for example, performed in fed-batch or perfusion mode toprepare for the standard industry fill-finish process in Step 7. FIG. 1Cshows an exemplary continuous process flow wherein Step 1 comprises abioreactor producing a biological product at steady-state, Step 2comprises a continuous dynamic filtration module, Step 3 comprises anaffinity-based, magnetic purification module, Step 4 comprises anisoelectric point-based, fluidic purification module, Step 5 comprises astandard industry viral inactivation and filtration process, forexample, performed in fed-batch or perfusion mode, and Step 6 compriseshigh performance tangential flow filtration with a charged membraneperformed in fed-batch or perfusion mode to prepare for the standardindustry fill-finish process in Step 7. FIG. 1D shows an exemplarycontinuous process flow wherein Step 1 comprises a bioreactor producinga biological product at steady-state, Step 2 comprises a continuousdynamic filtration module, Step 3 comprises an affinity-based, magneticpurification module, Step 4 comprises an isoelectric point-based,fluidic purification module, Step 5 comprises high performancetangential flow filtration with a charged membrane performed infed-batch or perfusion mode to prepare for the standard industryfill-finish process in Step 6.

FIGS. 2A-2D show schematics of exemplary continuous process flowsdescribed herein. FIG. 2A shows an exemplary continuous process flowwherein Step 1 comprises a bioreactor producing a biological product atsteady-state, Step 2 comprises a continuous dynamic filtration module,Step 3 comprises an affinity-based purification module, Step 4 comprisesat least one charge-based purification module, Step 5 comprises astandard industry viral inactivation and filtration process, forexample, performed in fed-batch or perfusion mode, and Step 6 compriseshigh performance tangential flow filtration with a charged membrane, forexample, performed in fed-batch or perfusion mode to prepare for thestandard industry fill-finish process in Step 7. FIG. 2B shows anexemplary continuous process flow wherein Step 1 comprises a bioreactorproducing a biological product at steady-state, Step 2 comprises acontinuous dynamic filtration module, Step 3 comprises an affinity-basedpurification module, Step 4 comprises a positive charge-basedpurification module, Step 5 comprises a negative charge-basedpurification module, and Step 6 comprises high performance tangentialflow filtration with a charged membrane, for example, performed infed-batch or perfusion mode to prepare for the standard industryfill-finish process in Step 7. FIG. 2C shows an exemplary continuousprocess flow wherein Step 1 comprises a bioreactor producing abiological product at steady-state, Step 2 comprises a continuousdynamic filtration module, Step 3 comprises an affinity-basedpurification module, Step 4 comprises an isoelectric point-based,fluidic purification module, Step 5 comprises a standard industry viralinactivation and filtration process, for example, performed in fed-batchor perfusion mode, and Step 6 comprises high performance tangential flowfiltration with a charged membrane performed in fed-batch or perfusionmode to prepare for the standard industry fill-finish process in Step 7.FIG. 2D shows an exemplary continuous process flow wherein Step 1comprises a bioreactor producing a biological product at steady-state,Step 2 comprises a continuous dynamic filtration module, Step 3comprises an affinity-based purification module, Step 4 comprises anisoelectric point-based, fluidic purification module, Step 5 compriseshigh performance tangential flow filtration with a charged membraneperformed in fed-batch or perfusion mode to prepare for the standardindustry fill-finish process in Step 6.

FIGS. 3A-3D show schematics of exemplary continuous process flowsdescribed herein. FIG. 3A shows an exemplary continuous process flowwherein Step 1 comprises a bioreactor producing a biological product atsteady-state, Step 2 comprises a continuous dynamic filtration module,Step 3 comprises an affinity-based, fluidic purification module, Step 4comprises at least one charge-based, fluidic purification module, Step 5comprises a standard industry viral inactivation and filtration process,for example, performed in fed-batch or perfusion mode, and Step 6comprises high performance tangential flow filtration with a chargedmembrane, for example, performed in fed-batch or perfusion mode toprepare for the standard industry fill-finish process in Step 7. FIG. 3Bshows an exemplary continuous process flow wherein Step 1 comprises abioreactor producing a biological product at steady-state, Step 2comprises a continuous dynamic filtration module, Step 3 comprises anaffinity-based, fluidic purification module, Step 4 comprises a positivecharge-based, fluidic purification module, Step 5 comprises a negativecharge-based, fluidic purification module, and Step 6 comprises highperformance tangential flow filtration with a charged membrane, forexample, performed in fed-batch or perfusion mode to prepare for thestandard industry fill-finish process in Step 7. FIG. 3C shows anexemplary continuous process flow wherein Step 1 comprises a bioreactorproducing a biological product at steady-state, Step 2 comprises acontinuous dynamic filtration module, Step 3 comprises anaffinity-based, fluidic purification module, Step 4 comprises anisoelectric point-based, fluidic purification module, Step 5 comprises astandard industry viral inactivation and filtration process, forexample, performed in fed-batch or perfusion mode, and Step 6 compriseshigh performance tangential flow filtration with a charged membraneperformed in fed-batch or perfusion mode to prepare for the standardindustry fill-finish process in Step 7. FIG. 3D shows an exemplarycontinuous process flow wherein Step 1 comprises a bioreactor producinga biological product at steady-state, Step 2 comprises a continuousdynamic filtration module, Step 3 comprises an affinity-based, fluidicpurification module, Step 4 comprises an isoelectric point-based,fluidic purification module, Step 5 comprises high performancetangential flow filtration with a charged membrane performed infed-batch or perfusion mode to prepare for the standard industryfill-finish process in Step 6.

FIGS. 4A-4D show schematics of exemplary continuous process flowsdescribed herein. FIG. 4A shows an exemplary continuous process flowwherein Step 1 comprises a bioreactor producing a biological product atsteady-state, Step 2 comprises a continuous dynamic filtration module,Step 3 comprises an affinity-based TFF purification module, Step 4comprises at least one charge-based TFF purification module, Step 5comprises a standard industry viral inactivation and filtration process,for example, performed in fed-batch or perfusion mode, and Step 6comprises high performance tangential flow filtration with a chargedmembrane, for example, performed in fed-batch or perfusion mode toprepare for the standard industry fill-finish process in Step 7. FIG. 4Bshows an exemplary continuous process flow wherein Step 1 comprises abioreactor producing a biological product at steady-state, Step 2comprises a continuous dynamic filtration module, Step 3 comprises anaffinity-based TFF purification module, Step 4 comprises a positivecharge-based TFF purification module, Step 5 comprises a negativecharge-based TFF purification module, and Step 6 comprises highperformance tangential flow filtration with a charged membrane, forexample, performed in fed-batch or perfusion mode to prepare for thestandard industry fill-finish process in Step 7. FIG. 4C shows anexemplary continuous process flow wherein Step 1 comprises a bioreactorproducing a biological product at steady-state, Step 2 comprises acontinuous dynamic filtration module, Step 3 comprises an affinity-basedTFF purification module, Step 4 comprises an isoelectric point-based,fluidic purification module, Step 5 comprises a standard industry viralinactivation and filtration process, for example, performed in fed-batchor perfusion mode, and Step 6 comprises high performance tangential flowfiltration with a charged membrane performed in fed-batch or perfusionmode to prepare for the standard industry fill-finish process in Step 7.FIG. 4D shows an exemplary continuous process flow wherein Step 1comprises a bioreactor producing a biological product at steady-state,Step 2 comprises a continuous dynamic filtration module, Step 3comprises an affinity-based TFF purification module, Step 4 comprises anisoelectric point-based, fluidic purification module, Step 5 compriseshigh performance tangential flow filtration with a charged membraneperformed in fed-batch or perfusion mode to prepare for the standardindustry fill-finish process in Step 6.

FIGS. 5A and 5B show an exemplary continuous process flow with designschematics for downstream purification modules described herein.

FIGS. 6A and 6B show a series of exemplary designs of the dynamicfiltration apparatus comprising a single output head to continuouslytransfer a heterogeneous mixture containing a biological product from asteady-state bioreactor bleed output line and a separate output head tosupply a wash buffer. FIG. 6A is a schematic of a dynamic filtrationapparatus design comprising a single output head to continuouslytransfer a heterogeneous mixture containing a biological product from asteady-state bioreactor bleed output line, a separate output head tosupply a wash buffer, a rolled filter membrane functioning as a supplyreel, a collection reel, two Servo motors to control the feedreel-to-collection reel system, two support rods having a mechanicallysmooth contact surface, a membrane support structure having amechanically smooth contact surface and an opening having continuitywith the vacuum line, a vacuum collection vessel, a diaphragm pump, anda peristaltic pump. FIG. 6B is a schematic of a dynamic filtrationapparatus design comprising a single output head to continuouslytransfer a heterogeneous mixture containing a biological product from asteady-state bioreactor bleed output line, a separate output head tosupply a wash buffer, a rolled filter membrane functioning as a feedreel, a collection reel, two Servo motors to control the feedreel-to-collection reel system, two support rods having a mechanicallysmooth contact surface, a membrane support structure having amechanically smooth contact surface and an opening having continuitywith the vacuum line, a controllable T-valve, two vacuum collectionvessels, a diaphragm pump, and two peristaltic pumps.

FIGS. 7A and 7B show two exemplary designs of the dynamic filtrationapparatus comprising multiple output heads to continuously transfer aheterogeneous mixture containing a biological product from asteady-state bioreactor bleed output line and multiple separate outputheads to supply a wash buffer. FIG. 7A depicts a schematic of a dynamicfiltration apparatus design comprising multiple output heads to transfera heterogeneous mixture containing a biological product from asteady-state bioreactor bleed output line, multiple separate outputheads to supply a wash buffer, a rolled filter membrane functioning as afeed reel, a collection reel, two Servo motors to control the feedreel-to-collection reel system, two support rods having a mechanicallysmooth contact surface, a membrane support structure having amechanically smooth contact surface and an opening having continuitywith the vacuum line, a vacuum collection vessel, a diaphragm pump, anda peristaltic pump. FIG. 7B depicts a schematic of a dynamic filtrationapparatus design comprising a multiple output heads to continuouslytransfer a heterogeneous mixture containing a biological product from asteady-state bioreactor bleed output line, multiple separate outputheads to supply a wash buffer, a rolled filter membrane functioning as afeed reel, a collection reel, two Servo motors to control the feedreel-to-collection reel system, two support rods having a mechanicallysmooth contact surface, a membrane support structure having amechanically smooth contact surface and an opening having continuitywith the vacuum line, a controllable T-valve, two vacuum collectionvessels, a diaphragm pump, and two peristaltic pumps.

FIG. 8 depict images of an exemplary membrane support structure havingan opening with five parallel slots.

FIGS. 9A and 9B are images showing the removal of PolyBeads (0.05%solids; 2 μm (red), 6 μm (red), and 10 μm (blue) diameter) from aheterogeneous mixture in 1×PBS. FIG. 9A is an image depicting the visualcomparison (left-to-right) of the initial heterogeneous mixture ofPolyBeads (0.05% solids; 2 μm, 6 μm, and 10μm diameter) in 1×PBS, thefiltrate resulting from purification of the heterogeneous mixture with a0.2μm PTFE syringe filter, the filtrate resulting from purification ofthe heterogeneous mixture with an exemplary dynamic filtration apparatus(MBM-3 represents a sample with a dynamic filtration with a 0.45μm PVDFfilter membrane and a flow rate of 0.25 mL/min), and the filtrateresulting from purification of the heterogeneous mixture with anexemplary dynamic filtration apparatus (MBM-4 represents a sample with adynamic filtration with a 0.45μm PES filter membrane and flow rates of0.25, 0.5, 1.0, 2.0, and 5.0 mL/min). FIG. 9B is a bar graph showing theUV-Vis spectrophotometric comparison of the initial heterogeneousmixture of PolyBeads (0.05% solids; 2 μm, 6 μm, and 10μm diameter) in1×PBS, the filtrate resulting from purification of the heterogeneousmixture with a 0.2μm PTFE syringe filter, the filtrate resulting frompurification of the heterogeneous mixture with an exemplary dynamicfiltration apparatus having a 0.45μm PVDF filter membrane and a flowrate of 0.25 mL/min (MBM-3), and the filtrate resulting frompurification of the heterogeneous mixture with an exemplary dynamicfiltration apparatus having a 0.45μm PES filter membrane and flow ratesof 0.25, 0.5, 1.0, 2.0, and 5.0 mL/min (MBM-4), thus demonstratingsuccessful removal of the PolyBeads from the heterogeneous mixture withthe exemplary dynamic filtration apparatus.

FIGS. 10A-10D are a series of data showing the removal of PolyBeads(0.05% solids; 2 μm (red), 6 μm (red), and 10μm (blue) diameter) from aheterogeneous mixture of PolyBeads suspended in a 0.5 mg/mL solution ofBSA-FITC in 1×PBS. FIG. 10A is a graph showing UV-Vis spectrophotometrictraces of a serially diluted heterogeneous mixture of PolyBeads (0.05%solids; 2 μm, 6 μm, and 10μm diameter) suspended in a 0.5 mg/mL solutionof BSA-FITC in 1×PBS showing the PolyBead signature region andindicating the presence of PolyBeads. FIG. 10B is a graph showing UV-Visspectrophotometric traces of a serially diluted 0.5 mg/mL solution ofBSA-FITC in 1×PBS showing the PolyBead signature region and indicatingthe absence of PolyBeads. FIG. 10C depicts an image of a visualcomparison (left-to-right) of the initial heterogeneous mixture ofPolyBeads (0.05% solids; 2 μm, 6 μm, and 10 μm diameter) suspended in a0.5 mg/mL solution of BSA-FITC in 1×PBS, the filtrate resulting frompurification of the heterogeneous mixture with a 0.2 μm PTFE syringefilter, the filtrate resulting from purification of the heterogeneousmixture with an exemplary dynamic filtration apparatus (MBM-3arepresents a sample with a dynamic filtration with a 0.45 μm PVDF filtermembrane and a flow rate of 0.25 mL/min), the filtrate resulting frompurification of the heterogeneous mixture with an exemplary dynamicfiltration apparatus (MBM-4a represents a sample with dynamic filtrationwith a 0.45 μm PES filter membrane and a flow rate of 0.5 mL/min), thefiltrate resulting from purification of the heterogeneous mixture withan exemplary dynamic filtration apparatus (MBM-5a represents a samplewith dynamic filtration with a 0.45 μm PES filter membrane and a flowrate of 2.0 mL/min), and the supernatant collected from purification ofthe heterogeneous mixture by centrifugation (5 min at 10,000×g). FIG.10D is a graph showing the UV-Vis spectrophotometric comparison of theinitial heterogeneous mixture of PolyBeads (0.05% solids; 2 μm, 6 μm,and 10 μm diameter) suspended in a 0.5 mg/mL solution of BSA-FITC in1×PBS, the filtrate resulting from purification of the heterogeneousmixture with a 0.2 μm PTFE syringe filter, the filtrate resulting frompurification of the heterogeneous mixture with an exemplary dynamicfiltration apparatus (MBM-3a), the filtrate resulting from purificationof the heterogeneous mixture with an exemplary dynamic filtrationapparatus (MBM-4a), the filtrate resulting from purification of theheterogeneous mixture with an exemplary dynamic filtration apparatus(MBM-5a), and the supernatant collected from purification bycentrifugation (5 min at 10,000×g), thus demonstrating successfulpurification of BSA-FITC by removal of the PolyBeads, as indicated bythe absence of PolyBeads in the PolyBead signature region, with theexemplary dynamic filtration apparatus.

FIGS. 11A and 11B show the dynamic filtration of a heterogeneous mixtureof PolyBeads suspended in a 0.5 mg/mL solution of human polyclonal IgG(hIgG) in 1×PBS at an input flow rate of 10 mL/min. FIG. 12A shows thedynamic filtration via a slot die output head. FIG. 11A depicts an imageof a visual comparison (left-to-right) of the initial heterogeneousmixture of PolyBeads (2 μm, 6 μm, and 10 μm diameter at 1.1×10⁸,4.2×10⁶, and 1.0×10⁶ particles/mL, respectively) suspended in a 0.5mg/mL solution of hIgG in 1×PBS, the filtrate resulting frompurification of the heterogeneous mixture with an exemplary dynamicfiltration apparatus having a 0.45 μm PES filter membrane and a flowrate of 10 mL/min, and the supernatant collected from purification ofthe heterogeneous mixture by centrifugation (5 min at 10,000×g). FIG.11B is a graph showing the spectrophotometric comparison of the totalprotein concentration as determined by BCA assay of the filtratesobtained by dynamic filtration and the supernatant collected bycentrifugation. FIG. 11C depicts an image of a visual comparison(left-to-right) of the initial heterogeneous mixture of PolyBeads (0.5μm, 0.75 μm, 1 μm, 2 μm, 3 μm, and 10 μm diameter at 7.3×10⁷, 1.1×10⁸,1.1×10⁸, 1.1×10⁸, 3.4×10⁷, and 1.0×10⁶ particles/mL, respectively)suspended in a 0.5 mg/mL solution of hIgG in 1×PBS, the filtrateresulting from purification of the heterogeneous mixture with anexemplary dynamic filtration apparatus having a 0.45 μm PES filtermembrane and a flow rate of 10 mL/min, and the supernatant collectedfrom purification of the heterogeneous mixture by centrifugation (5 minat 10,000×g). FIG. 11D is a graph showing the spectrophotometriccomparison of the total protein concentration as determined by BCA assayof the filtrates obtained by dynamic filtration and the supernatantcollected by centrifugation.

FIGS. 12A-12D show the protein recovery of dynamic filtration acrossdifferent proteins, protein concentrations, filter membrane materialsand pore sizes, and membrane support structures and during continuousoperation at different input flow rates. FIG. 12A shows the recovery ofproteins of different size and charge (bovine serum albumin (BSA),Lysozyme, and hIgG at 0.5-10 mg/mL, 5 mg/mL, and 0.5 mg/mL,respectively) by BCA assay of the filtrates obtained by dynamicfiltration with a 0.45 μm PES filter membrane having a 0.5 mm/sectransport velocity and an input flow rate of 10 mL/min. FIG. 12B showsthe recovery of hIgG at 0.5 mg/mL by BCA assay of the filtrates obtainedby dynamic filtration with filter membranes of different materials andpore sizes (0.45 μm PES, 0.45 μm hydrophilic PVDF, 0.22 μm PES) having a0.5 mm/sec transport velocity and an input flow rate of 10 mL/min. FIG.12C shows the recovery of hIgG at 0.5 mg/mL by BCA assay of thefiltrates obtained by dynamic filtration with different membrane supportstructures (a PTFE membrane support structure with 5 parallel slots anda PTFE membrane support structure with a porous hydrophilic polyethylene(PE) insert) and a 0.45 μm PES filter membrane having a 0.5 mm/sectransport velocity and an input flow rate of 10 mL/min. FIG. 12D showsthe recovery of Lysozyme at 0.5 mg/mL by BCA assay of the filtratesobtained by dynamic filtration at different flow rates (5 and 10 mL/min)during long-term, continuous operation with a 0.45 μm PES filtermembrane having a 0.5 mm/sec transport velocity.

FIGS. 13A-13C show the comparison of cell clarification by dynamicfiltration and centrifugation of an input heterogeneous mixturecomprising a suspension cell culture in RPMI media spiked with hIgG to afinal concentration of 1 g/L. FIG. 13A shows optical imaging(left-to-right) of the initial hIgG-spiked, murine myeloma suspensioncell culture in RPMI media (1 g/L hIgG in 2×10⁶ cells/mL), filtrates(DF-1, DF-2, DF-3) obtained by dynamic filtration at an input flow rateof 2 mL/min with a 0.45 μm PES filter membrane having a transportvelocity of 0.5 mm/sec, and supernatants (C-1, C-2, C-3) collected fromcentrifugation for 5 minutes at 10,000×g. FIG. 13B shows SDS-PAGEanalysis (left-to-right) of the initial hIgG-spiked, murine myelomasuspension cell culture in RPMI media (1 g/L hIgG in 2×10⁶ cells/mL),filtrates (DF-1, DF-2, DF-3) derived from dynamic filtration at an inputflow rate of 2 mL/min with a 0.45 μm PES filter membrane having atransport velocity of 0.5 mm/sec, and supernatants (C-1, C-2, C-3)derived from centrifugation for 5 minutes at 10,000×g. FIG. 13C showsthe comparison of the recovery of hIgG from the heterogeneous mixture(suspension cell culture in RPMI media spiked with 1 g/L hIgG) by BCAassay of the filtrates obtained by dynamic filtration at an input flowrate of 2 mL/min with a 0.45 μm PES filter membrane having a transportvelocity of 0.5 mm/sec (blue outlined bar) and the supernatantscollected from centrifugation for 5 minutes at 10,000×g.

FIG. 14 shows an exemplary design schematic of an affinity-based,magnetic purification apparatus comprising a loop conveyor system and atleast one magnetic field that is permanently “on”.

FIG. 15 shows an exemplary design schematic of an affinity-based,magnetic purification apparatus comprising a loop conveyor system and atleast one magnetic field capable of “on/off” toggling.

FIG. 16 shows an exemplary design schematic of a charge-based, magneticpurification apparatus comprising a loop conveyor system and at leastone magnetic field that is permanently “on”.

FIG. 17 shows an exemplary design schematic of a charge-based, magneticpurification apparatus comprising a loop conveyor system and at leastone magnetic field capable of “on/off” toggling.

FIG. 18 shows an exemplary design schematic of an affinity-based,magnetic purification apparatus comprising a pick and place roboticssystem and at least one magnetic field.

FIG. 19 shows an exemplary design schematic of a charge-based, magneticpurification apparatus comprising a pick and place robotics system andat least one magnetic field.

FIGS. 20A and 20B show the affinity-based, magnetic purification of amixture of hIgG (target, 2 g/L input concentration) and Lysozyme(impurity, 1 g/L input concentration) performed with an affinity-based,magnetic purification apparatus charged with magnetic, Protein A-coatedagarose beads. FIG. 20A shows total protein analysis by BCA assay acrossfractions collected from 3 sequential cycles of magnetic affinity beaduse and recycling demonstrating the ability to reproducibly recycle andreuse the magnetic affinity beads without compromising binding capacityand performance. FIG. 20B shows SDS-PAGE analysis across fractionscollected from 3 sequential cycles of magnetic affinity bead use andrecycling demonstrating the ability to reproducibly recycle and reusethe magnetic affinity beads without compromising binding capacity andperformance.

FIGS. 21A-21D show an exemplary design of an affinity-based purificationapparatus comprising a mechanical rotary system. FIG. 21A is a schematicof a lid system. FIG. 21B is a schematic of a vessel carousel. FIG. 21Cis a schematic of a collection system. FIG. 21D depicts the manner towhich the lid system and collection system interface with the vesselcarousel.

FIGS. 22A-22D show an exemplary design of a charge-based purificationapparatus comprising a mechanical rotary system. FIG. 22A is a schematicof a lid system. FIG. 22B is a schematic of a vessel carousel. FIG. 22Cis a schematic of a collection system. FIG. 22D depicts the manner towhich the lid system and collection system interface with the vesselcarousel.

FIGS. 23A-23D show the individual system components of an affinity-basedpurification or a charge-based purification apparatus. FIG. 23A is aschematic of an exemplary gasketed lid, vessel, and collector assembly.FIG. 23B is a schematic of an exemplary gasketed lid comprising an airinlet, two buffer inlets configured to generate a circular flow pattern,a vent port, and a filling inlet that is a component of the lid system.FIG. 23C is a schematic of a vessel comprising, a mesh filter or a frit,and a valve that is a component of the vessel carousel. FIG. 23D is aschematic of the collector that is a component of the collection system.

FIGS. 24A and 24B show an exemplary design of an affinity-basedpurification apparatus comprising a staged linear system. FIG. 24A showthe individual system components of an affinity-based purificationapparatus. FIG. 24B show connectivity of the affinity-based purificationapparatus comprising a staged linear system.

FIGS. 25A and 25B show an exemplary design of an affinity-basedpurification apparatus comprising a staged linear system. FIG. 24A showthe individual system components of a charge-based purificationapparatus. FIG. 24B show connectivity of the charge-based purificationapparatus comprising a staged linear system.

FIGS. 26A and 26B shows the affinity-based purification of a solution ofhIgG (2 g/L input concentration) performed with an affinity-basedpurification apparatus charged with Protein A-coated agarose resinbeads. FIG. 26A shows total protein analysis by BCA assay acrossfractions collected from 3 sequential cycles of affinity resin bead useand recycling demonstrating the ability to reproducibly recycle andreuse the affinity resin beads without compromising binding capacity andperformance. FIG. 26B shows SDS-PAGE analysis across fractions collectedfrom 3 sequential cycles of affinity resin bead use and recyclingdemonstrating the ability to reproducibly recycle and reuse the affinityresin beads without compromising binding capacity and performance.

FIGS. 27A and 27B shows the affinity-based purification of a mixture ofhIgG (target, 2 g/L input concentration) and Lysozyme (impurity, 1 g/Linput concentration) performed with an affinity-based purificationapparatus charged Protein A-coated agarose resin beads. FIG. 27A showstotal protein analysis by BCA assay across fractions collected from 3sequential cycles of affinity resin bead use and recycling demonstratingthe ability to reproducibly recycle and reuse the affinity resin beadswithout compromising binding capacity and performance. FIG. 27B showsSDS-PAGE analysis across fractions collected from 3 sequential cycles ofaffinity resin bead use and recycling demonstrating the ability toreproducibly recycle and reuse the affinity resin beads withoutcompromising binding capacity and performance.

FIGS. 28A-28D are a series of images showing exemplary designs of hybridfluidic devices. FIG. 28A is an image showing a hybrid fluidic devicecomprising a parallel cross-flow channel, a permanent magnetic field,and two piezoelectric transducers. FIG. 28B is an image showing a hybridfluidic device comprising an angled cross-flow channel, a permanentmagnetic field, and two piezoelectric transducers. FIG. 28C is an imageshowing a hybrid fluidic device comprising a parallel cross-flowchannel, a permanent magnetic field, and two selective dielectrophoreticelectrodes. FIG. 28D is an image showing a hybrid fluidic devicecomprising an angled cross-flow channel, a permanent magnetic field, andtwo selective dielectrophoretic electrodes.

FIG. 29 shows an exemplary design schematic of an affinity-based,fluidic purification apparatus.

FIG. 30 shows an exemplary design schematic of a charge-based, fluidicpurification apparatus.

FIG. 31 shows an exemplary design schematic of an affinity-basedpurification apparatus comprising at least one tangential flowfiltration systems.

FIG. 32 shows an exemplary design schematic of a charge-basedpurification apparatus comprising at least one tangential flowfiltration systems.

FIG. 33 shows an exemplary design schematic of an isoelectricpoint-based, fluidic purification apparatus comprising a fluidic devicehaving a channel created between two parallel plates, an electric fieldorthogonal to the direction of the fluid flow, and an aqueous ionicsolution.

FIG. 34 shows an exemplary design schematic of a free-flowelectrophoresis apparatus comprising a first fluidic device having achannel created between two parallel plates, an electric fieldorthogonal to the direction of the fluid flow, and a coarse pH gradient,and that is connected to a second fluidic device having a channelcreated between two parallel plates, an electric field orthogonal to thedirection of the fluid flow, and a fine pH gradient, wherein theapparatus is capable of operating in isoelectric focusing mode.

FIG. 35 shows an exemplary design schematic of a free-flowelectrophoresis apparatus comprising a first fluidic device having achannel created between two parallel plates, an electric fieldorthogonal to the direction of the fluid flow, and a constant basic pHacross the main separation channel, that is connected to a secondfluidic device having a channel created between two parallel plates, anelectric field orthogonal to the direction of the fluid flow, and aconstant acidic pH across the main separation channel, wherein theapparatus is capable of operating in zone electrophoresis mode.

FIG. 36 shows an exemplary design schematic of a free-flowelectrophoresis apparatus comprising a first fluidic device having achannel created between two parallel plates and an electric fieldorthogonal to the direction of the fluid flow capable of operating inisoelectric focusing mode that is connected to a second fluidic devicehaving a channel created between two parallel plates and an electricfield orthogonal to the direction of the fluid flow capable of operatingin isotachophoresis mode.

FIG. 37 shows an exemplary design schematic of a free-flowelectrophoresis apparatus comprising a first fluidic device having achannel with a selective dielectrophoretic electrode to pre-sort amixture that is connected to a second fluidic device having a channelcreated between two parallel plates and an electric field orthogonal tothe direction of the fluid flow capable of operating in isoelectricfocusing mode that is connected to a third fluidic device having achannel created between two parallel plates and an electric fieldorthogonal to the direction of the fluid flow capable of operating inisotachophoresis mode.

FIG. 38 shows the design of an exemplary de-bubbling and de-gassingsystem that removes electrolysis bubbles directly from the electrodechannels to create a bubble-free main separation channel.

FIG. 39 shows an exemplary liquid circuit breaker that creates a breakin the solution connected to applied voltage flowing from the outlet ofa free-flow electrophoresis apparatus to at least one in-line sensor ordetector.

FIGS. 40A-40E show isoelectric point-based, fluidic purification of amixture of Rhodamine 6G (0.25 mg/mL, net charge of +1) and Fluorescein(0.25 mg/mL, net charge of −1) with an isoelectric point-basedpurification apparatus with an anodic channel (H₂SO₄), a cathodicchannel (NaOH), and a main separation channel having five inlets andfive outlets flowing an ampholyte solution, wherein the mixture wasintroduced at the center of the apparatus' inlets (inlet 3). FIG. 40Ashows an optical image of the fractions collected from the five outletsat 0V and 5 mL/min. FIG. 40B shows the absorbance spectra of thefractions collected from the five outlets at 0V and 5 mL/min. FIG. 40Cshows an optical image of the fractions collected from the five outletsat 1000V and 10 mL/min in the presence of a pH gradient. FIG. 40D showsthe absorbance spectra of the fractions collected from the five outletsat 1000V and 10 mL/min in the presence of a pH gradient. FIG. 40E showsthe purification of the mixture resulting in fractions containingpurified Rhodamine 6G (outlet 2, towards the cathode) and purifiedFluorescein (outlet 4, toward the anode).

FIGS. 41A-41C show optical imaging of the purification of a mixture ofRhodamine 6G (0.25 mg/mL, net charge of +1) and Fluorescein (0.25 mg/mL,net charge of −1) under different operating conditions with anisoelectric point-based purification apparatus with an anodic channel(H₂SO₄), a cathodic channel (NaOH), and a main separation channel havingfive inlets and five outlets flowing an ampholyte solution, wherein themixture was introduced at the center of the apparatus' inlets (inlet 3).FIG. 41A shows isoelectric focusing free-flow electrophoresis of amixture of Rhodamine 6G and Fluorescein at 500V and a flow rate of 3mL/min. FIG. 41B shows isoelectric focusing free-flow electrophoresis ofa mixture of Rhodamine 6G and Fluorescein at 700V and a flow rate of 5mL/min. FIG. 41C shows isoelectric focusing free-flow electrophoresis ofa mixture of Rhodamine 6G and Fluorescein at 900V and a flow rate of 10mL/min.

FIGS. 42A and 42B show optical imaging of the purification of a mixtureof small-molecule dyes with an isoelectric point-based purificationapparatus with an apparatus comprising anodic channel (H₂SO₄), acathodic channel (NaOH), and a main separation channel having fiveinlets and five outlets flowing an ampholyte solution, wherein themixture was introduced at the center of the apparatus' inlets (inlet 3).FIG. 42A shows isoelectric focusing free-flow electrophoresis of amixture of Basic Fuchsin (0.05 mg/mL, net charge of +3) and Fluorescein(0.25 mg/mL, net charge of −1) at 500V and a flow rate of 5 mL/min. FIG.42B shows isoelectric focusing free-flow electrophoresis of a mixture ofCrystal Violet (0.05 mg/mL, net charge of +3) and Fluorescein (0.25mg/mL, net charge of −1) at 500V and a flow rate of 5 mL/min.

FIGS. 43A-43D show optical imaging of the purification of a mixture ofBasic Fuchsin (0.005 mg/mL, net charge of +3) and Fluorescein (0.25mg/mL, net charge of −1) with an isoelectric point-based purificationapparatus operating in an isoelectric focusing free-flow electrophoresismode across increasing applied voltages. The mixture was introduced intothe central inlet (inlet 3) at a flow rate of 5 mL/min with an apparatuscomprising an anodic channel, a cathodic channel, and a main separationchannel having five inlets and ten outlets, wherein each channel wasflowing the same ampholyte solution at 5 mL/min. When no voltage isapplied, the mixture follows laminar flow and exits the apparatus at thecentral outlets (outlets 4 and 5) (FIG. 43A). When voltage is appliedacross the main separation channel having an ampholyte and sample inputflow rate of 5 mL/min, a linear pH gradient is established and BasicFuchsin and Fluorescein migrate to the cathode and anode, respectively,consistent with theoretical electrophoretic mobility predictions (FIGS.43B-43D). As the applied voltage was increased from 600V (FIG. 43B), to900V (FIG. 43C) to 1100V (FIG. 43D) to generate an increase in theE-field strength, the separation of the two molecules was observed toproportionally increase over the length of the main separation channel.

FIGS. 44A and 44B show optical imaging of the purification of a mixtureof small-molecule dyes with an isoelectric point-based purificationapparatus with an apparatus comprising anodic channel (H₂SO₄), acathodic channel (NaOH), and a main separation channel having fiveinlets and five outlets flowing two ampholyte solutions separated by aspacer solution, wherein the mixture was introduced in the spacersolution at the center of the apparatus' inlets (inlet 3). FIG. 44Ashows isotachophoresis of a mixture of Rhodamine 6G (0.25 mg/mL, netcharge of +1) and Fluorescein (0.25 mg/mL, net charge of −1) at 250V anda flow rate of 5 mL/min resulting in concentration of the two dyes intotwo discrete high resolution lines. FIG. 44B shows UV illumination ofthe results presented in FIG. 46A.

FIGS. 45A-45E show the purification of a mixture of BSA (0.5 mg/mL, pIof 4-5) and Lysozyme (0.25 mg/mL, pI of 11) with an isoelectricpoint-based purification apparatus with an apparatus comprising anodicchannel (H₂SO₄), a cathodic channel (NaOH), and a main separationchannel having five inlets and five outlets flowing an ampholytesolution, wherein the mixture was introduced at the center of theapparatus' inlets (inlet 3). FIG. 45A shows the spectrophotometricanalysis by BCA assay of the total protein concentration of thefractions derived from the five outlets at 0V and a flow rate of 10mL/min. FIG. 45B shows the SDS-PAGE analysis of the fractions derivedfrom the five outlets at 0V and a flow rate of 10 mL/min, showing themixture present in outlet 3. FIG. 45C shows the spectrophotometricanalysis by BCA assay of the total protein concentration of thefractions derived from the five outlets at 850V and a flow rate of 10mL/min, showing a distribution of protein across outlets 2, 3, and 4.FIG. 45D shows the SDS-PAGE analysis of the fractions derived from thefive outlets at 850V and a flow rate of 10 mL/min, showing the presenceof purified Lysozyme in outlet 2 and purified BSA in outlet 4. FIG. 45Eshows the theoretical electrophoretic migration direction of BSA (towardthe anode) and Lysozyme (toward the cathode).

FIGS. 46A-46C show the purification of a mixture of hIgG (0.5 mg/mL, pIof 6-8) and Lysozyme (0.25 mg/mL, pI of 11) with an isoelectricpoint-based purification apparatus with an apparatus comprising anodicchannel (H₂SO₄), a cathodic channel (NaOH), and a main separationchannel having five inlets and five outlets flowing an ampholytesolution, wherein the mixture was introduced at the center of theapparatus' inlets (inlet 3). FIG. 46A shows the spectrophotometricanalysis by BCA assay of the total protein concentration of thefractions derived from the five outlets at either (1) 0V, 5 mL/min, (2)1000V, 5 mL/min, (3) 1500V, 5 mL/min, (4) 0V, 10 mL/min, or (5) 1000V,10 mL/min. FIG. 46B shows the theoretical electrophoretic migrationdirection of hIgG (toward the anode, cathode and center) and Lysozyme(toward the cathode). FIG. 46C shows the SDS-PAGE analysis of thefractions derived from the five outlets at either (1) 0V, 5 mL/min, (2)1000V, 5 mL/min, (3) 1500V, 5 mL/min, (4) 0V, 10 mL/min, or (5) 1000V,10 mL/min.

FIGS. 47A-47D show the purification of a mixture of BSA (0.5 mg/mL, pIof 4-5) and Lysozyme (0.25 mg/mL, pI of 11) with an isoelectricpoint-based purification apparatus with an apparatus comprising anodicchannel (H₂SO₄), a cathodic channel (NaOH), and a main separationchannel having five inlets and five outlets flowing an ampholytesolution, wherein the mixture was introduced at the center of theapparatus' inlets (inlet 3). FIG. 47A shows the spectrophotometricanalysis by BCA assay of the total protein concentration of thefractions derived from the five outlets at 0V and 500V at a flow rate of3 mL/min, showing a distribution of protein across outlets 2, 3, and 4under applied voltage. FIG. 47B shows the spectrophotometric analysis byBCA assay of the total protein concentration of the fractions derivedfrom the five outlets at 0V and 700V at a flow rate of 5 mL/min, showinga distribution of protein across outlets 2, 3, and 4 under appliedvoltage. FIG. 47C shows the spectrophotometric analysis by BCA assay ofthe total protein concentration of the fractions derived from the fiveoutlets at 0V and 850V at a flow rate of 10 mL/min, showing adistribution of protein across outlets 2, 3, and 4 under appliedvoltage. FIG. 47D shows the SDS-PAGE analysis of the fractions derivedfrom the five outlets at either (1) 0V or 500V at 3 mL/min, (2) 0V or700V at 5 mL/min, or (3) 0V or 850V at 10 mL/min.

FIG. 48 shows an exemplary a schematic of connecting a charge-based,magnetic, a charge-based, a charge-based fluidic, a charge-based TFF, oran isoelectric point-based purification module to an exemplarysemi-continuous process, described herein, utilizing standard industrydownstream processing equipment run in fed-batch or perfusion mode toprepare the biological product for fill-finish.

FIG. 49 shows an exemplary a schematic of connecting a charge-based,magnetic, a charge-based, a charge-based fluidic, a charge-based TFF, oran isoelectric point-based purification module to an exemplarysemi-continuous process, described herein, utilizing standard industrydownstream processing equipment run in fed-batch or perfusion mode toprepare the biological product for fill-finish in the absence of anindependent viral inactivation and removal process step.

DETAILED DESCRIPTION

Provided herein is, inter alia, a continuous process for purifying abiological product. The presently claimed process provides for a numberof advantages over current downstream methods and processes forpurifying a biological product, for example, a protein or fragmentthereof (a polypeptide) an antibody or fragment thereof, a cytokine, achemokine, an enzyme, a growth factor, an oligonucleotide, a virus, anadenovirus, an adeno-associated virus, or a lentivirus. For example,without intent to be limiting, the process described herein provides acontinuous bioprocess for purifying a monoclonal antibody that abrogatesthe problem of membrane fouling inherent to traditional multiple stagefiltration processes (e.g. multiple stage tangential flow filtration ordepth filtration) by having the initial stage of filtration comprise atleast one dynamic filtration module, as described herein, to removelarge impurities (e.g. cells, cellular debris, and aggregates). Further,the continuous process maintains throughput and yield, whilesignificantly decreasing the production facility footprint, the timerequired for facility buildout and validation, the costs associated withfacility buildout, and capital equipment expenditure, when compared tothe traditional approaches of batch, single-use, or semi-continuousmonoclonal antibody manufacturing.

The continuous bioprocessing as described herein affords smaller, morestreamlined equipment (e.g., smaller bioreactor volumes and downstreambioprocess equipment) because the ability to operate continuouslyeliminates the need for the large process equipment required for thecentrifugation, depth filtration, and column chromatography steps oftraditional downstream bioprocessing, whose size is dictated by largebioreactor volumes. Further, the smaller, more streamlined equipmentoperating continuously affords the use of significantly smallerbioreactor(s) that produces monoclonal antibodies at steady-state. Thecontinuous bioprocess as described herein may also significantlydecrease operating expenditures, overall bioprocess line downtime, andbiological product loss when compared to traditional monoclonal antibodymanufacturing approaches. Finally, the process described herein forpurifying a biological product is conducted in a system with a footprintthat occupies significantly less square footage than current techniques,without sacrificing product throughput or yield on a kilogram/yearbasis. For example, the process for producing, purifying a monoclonalantibody as described herein is operated with a footprint that occupiesup to about 30,000 square feet. In contrast, current monoclonal antibodyproduction and downstream processes require at least 200,000 squarefeet.

Continuous Process for Purifying a Biological Product Using a DynamicFiltration Module, an Affinity-Based, Magnetic Purification Module, andat Least One of a Charge-Based, Magnetic Purification Module or anIsoelectric Point-Based, Fluidic Purification Module.

A continuous process for purifying a biological product is described;the process including continuously receiving, via an input line, aheterogeneous mixture containing the biological product, wherein thebiological product includes, but is not limited to, a protein orfragment thereof (a polypeptide), an antibody or fragment thereof, acytokine, a chemokine, an enzyme, or a growth factor. When purifying,the biological product (e.g., a monoclonal antibody) is substantiallypure when it is at least 60%, 70%, 80%, 90%, 95%, or even 99%, byweight, free from impurities (cells, cellular debris, aggregates, hostcell proteins, undesired proteins and peptides, undesired antibodies,undesired nucleic acids and oligonucleotides, viruses, salts, buffercomponents, surfactants, sugars, metallic contaminants, leachables,media components, and/or naturally-occurring organic molecules withwhich it is naturally associated).

The process includes continuously removing large impurities from theheterogeneous mixture by dynamic filtration. Said dynamic filtrationprocess includes at least one dynamic filtration module thatcontinuously feeds the biological product from at least one output headin fluid communication with the input line to the dynamic filtrationmodule under negative pressure, thereby producing a filtrate comprisingthe biological product. The dynamic filtration module may furtherinclude at least one additional input line to supply a wash buffer via acoaxial output head or a separate monoaxial output head.

In embodiments, the process described herein includes purifying abiological product that is continuously produced in a bioreactor (e.g.,a fed-batch bioreactor, a perfusion bioreactor, a chemostat bioreactor).For example, the bioreactor includes a bioreactor feed line and anoutput bleed line to enable steady-state cell culture growth conditions,and the output bleed line functions as the input line to permitcontinuous fluid flow from the bioreactor to the dynamic filtrationmodule.

As described herein, the process of continuously removing largeimpurities from the heterogeneous mixture (or mixture) does not includecentrifugation, disk-stack centrifugation, depth filtration, staticfiltration, tangential flow filtration, a hydrocyclone, or anycombination thereof. The term “static filtration” refers to a process inwhich the heterogeneous mixture being filtered remains static, meaning,for example, that the filter membrane (or depth filter) has a definedcapacity, and the rate of filtration decreases as the membrane reachesits capacity (e.g., membrane pores become occluded). In a “static” (asopposed to “dynamic”) filtration, the filter membrane remains stationary(does not move), and the flow (e.g., of the heterogeneous mixture)passes through the stationary filter membrane. These static filtrationmethods are common in the art and are simple and well-understood.

Unlike the static filtration methods commonly used in the art, theprocess herein describes a dynamic filtration module, wherein componentsof the dynamic filtration module move in a coordinated fashion (e.g.,the membrane moves or advances in accordance with the flow rate of theentire process) to enable filtration to occur continuously across afresh, unused target region of filter membrane. This eliminates membranefouling or occlusion and permits control over the filter cake packingand thickness during operation.

The dynamic filtration module includes a filter membrane roll, amembrane support structure, at least one support rod or roller, a vacuumline, a vacuum system, and at least one vacuum collection vessel.

In embodiments, the filter membrane roll includes a rolled filtermembrane, wherein the filter membrane, without intent to be limiting,comprises polyethersulfone (PES), hydrophilic polysulfone, celluloseester, cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilicPVDF, polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilicPTFE, or any combination thereof.

The pore size of the rolled filter membrane depends on the biologicalproduct being purified. In examples, the rolled filter membrane has apore size in the range from 0.1 μm to 1 μm. Alternatively, the pore sizeis in the range from about 0.2 μm to about 0.45 μm, or the pore size isless than about 0.45 μm. In other examples, when purifying an antibody,the pore size of the rolled filter membrane is in the range of 0.2 μm toabout 0.45μm.

The filter membrane roll has a width from about 10 mm to about 600 mm.The width of the filter membrane roll, for example, may depend on thesize of the dynamic filtration system or the membrane support structure.

In embodiments, the filter membrane roll further functions as a feedreel that communicates with a collection reel, meaning the filtermembrane originates from pre-fabricated roll and spans to an initiallyempty collecting roll, thus creating a reel-to-reel system. In aspects,the dynamic filtration module includes a rolled filter membraneextending between a feed reel and a collection reel, the filter membranehaving a target region (e.g., an active target region) that isconfigured to receive the heterogeneous mixture. In examples, the feedreel motion is governed by a Servo motor coupled with a gear box tolimit rotations per minute (RPM) by a ratio of 200:1 to enable lowmembrane transport velocities with high torque. The collection reelmotion is governed by a Servo motor coupled with a gear box to limit RPMby a ratio of 200:1 to enable low membrane transport velocities withhigh torque. Further, the feed reel motor and the collection reel motorare controlled by a closed-loop controller that operates a feedbackmechanism to ensure consistent velocity with the constantly changingdiameters of the filter membrane roll on both the feed reel and thecollection reel during operation. In examples, the feed reel and thecollection reel operate in the same direction with equivalentvelocities.

In embodiments, the transport velocity of the filter membrane rangesfrom about 0.1 mm/sec to about 100 mm/sec, preferably from about 0.1mm/sec to about 10 mm/sec.

The membrane support structure of the dynamic filtration module includesa mechanically smooth contact surface derived from a material having alow static coefficient of friction (e.g. PTFE) and an opening that hascontinuity with the vacuum line. As used herein, the “membrane supportstructure” refers to a fabricated component that provides structuralsupport to the active region of the filter membrane, to preventdeformation, as it traverses an area of negative pressure, resultingfrom the opening having continuity with the vacuum line. Further, asused herein, “mechanically smooth contact surface” refers to a surfacehaving a low static coefficient of friction, thus creating a lowfrictional force opposing transport of the filter membrane, especiallywhen wetted. The mechanically smooth contact surface may influence theease at which the filter membrane moves in a dynamic fashion. Themechanically smooth contact surface may also be measured in surfaceroughness, where the lower the value the smoother the surface. Moreover,since rougher surfaces have more friction between them than smoothersurfaces, the mechanically smooth contact surface, as used herein,refers to a surface having lower friction (i.e., a low staticcoefficient of friction).

In embodiments, the membrane support structure of the dynamic filtrationmodule includes an opening. The opening for example, may include a mesh,at least one slot, at least one hole, a frit, a porous material, or anycombination thereof. For example, the opening may include a series ofregularly or irregularly spaced elements (e.g., a mesh, at least oneslot, at least one hole, or any combination thereof). Moreover, theopening may include regularly spaced elements, for example the openingmay include a series of equally spaced, parallel slots. Additionally,the opening can include one grate (e.g., a series of regularly orirregularly spaced elements as described above). In other examples, theopening can include more than one grate, with each grate perpendicular.The opening can be a collection of irregular or regular elements (e.g.,a series of parallel slots). The opening can also include a mesh, whichare of split-thickness or of full-thickness and may or may not be inparallel rows. The elements of the opening (e.g., a mesh, at least oneslot, at least one hole, a frit, a porous material, or any combinationthereof) may be of any desired thickness. For example, without intent tobe limiting, the opening may include a mesh with a thickness of about0.25 mm to about 5 mm.

The membrane support structure of the dynamic filtration module includesa temperature control mechanism. The temperature control mechanismmaintains a temperature from about 4° C. to about 37° C. in the presenceof evaporative cooling. For example, during purification of an antibody,the temperature control mechanism maintains a temperature from 15° C. to37° C. Exemplary temperature control mechanisms include, but are notlimited to, single loop controllers, multi-loop controllers, closed loopcontrollers, PID controllers, Peltier devices, resistive heatingelements, and/or thermal chucks with circulating water/propylene glycoljackets.

In embodiments, the at least one support rod or roller of the dynamicfiltration module has a mechanically smooth contact surface derived froma material having a low static coefficient of friction (e.g. PTFE, PFA).For example, the static coefficient of friction ranges from about 0.01to about 0.1, or from about 0.01 to about 0.05, or from about 0.05 toabout 0.1. In specific examples, the static coefficient of friction is0.04. In examples, the dynamic filtration module includes at least onesupport rod or roller with a mechanically smooth contact surface tostabilize the motion of the filter membrane across the membrane supportstructure.

In embodiments, the dynamic filtration module includes at least oneoutput head for modulating flow of the heterogeneous mixture anddispensing the heterogeneous mixture onto the target region (e.g., anactive target region) of the filter membrane. In examples, the at leastone output head is a tube or a slot die.

In some embodiments, the dynamic filtration module further includes atleast one additional input line to supply a wash buffer via a coaxialoutput head, a separate monoaxial output head, a separate slot dieoutput head, or a slot die output head with multiple openings.

In some embodiments, the dynamic filtration module includes elementsknown in the coating and converting industry, for example, withoutintent to be limiting, active or passive edge guides, tension control(e.g. a dancer), break and tension detectors, or any combinationthereof.

In embodiments, the dynamic filtration module includes a vacuum systemhaving continuity with the membrane support structure to apply negativepressure across the active target region of the filter membrane, wherethe negative pressure allows for active transport of the filter membraneacross the membrane support structure and enables collection of thefiltrate containing the biological product. In examples, the vacuumsystem of the dynamic filtration module maintains a gauge pressure ofabout −0.05 bar to about 0.98 bar for continuous filtration.

In embodiments, the dynamic filtration module further includes at leastone vacuum collection vessel configured to collect the filtrate, and atleast one sensor or detector. In aspects described herein, during thepurification by dynamic filtration, the filtrate comprising thebiological product is fed under negative pressure into a vacuumcollection vessel capable of collecting from about 50 mL to about 100 L.In examples, the vacuum collection vessel capable of collecting thefiltrate is from about 1 L to about 10 L. In other examples, the vacuumcollection vessel capable of collecting the filtrate is from about 1 Lto about 50 L.

In embodiments, the process of continuously removing large impurities(e.g., cells, cell debris, and aggregates) from the heterogeneousmixture by dynamic filtration comprises a multiple stage filtration withat least two discrete rolled filter membranes with different pore sizes.In examples, this multiple stage dynamic filtration process includes atleast one first dynamic filtration apparatus having a rolled filtermembrane with a large pore size (e.g., 0.45 μm) in fluid communicationwith at least one second dynamic filtration apparatus having a rolledfilter membrane with a small pore size (e.g., 0.2 μm), thereby producinga filtrate comprising the biological product.

The process described herein includes continuously transferring thefiltrate to a first module capable of separating the solution into twoor more fractions comprising at least one fraction containing thebiological product. For example, separating the solution into two ormore fractions, may include at least one fraction containing thebiological product, and the at least one other fraction containing smallimpurities. As described herein, the first module comprises anaffinity-based, magnetic purification apparatus. The “affinity-based,magnetic purification apparatus” refers to a purification techniquebased upon molecular, conformational binding interactions (e.g.,ligand-receptor interactions) in which selective surface-immobilizedligands recognize and bind to the biological product to be purified. Inexamples, the first module has at least one first inlet and at least onefirst outlet and is configured to permit continuous fluid flow betweenthe first inlet and the first outlet via a loop conveyor system or apick and place robotics system.

In embodiments, the affinity-based, magnetic purification apparatusfurther includes a suspension of magnetic resin beads. The surface ofthe magnetic resin beads, for example, without intent to be limiting, iscoupled with Protein A, Protein G, Protein L, an antigenic protein, aprotein, a receptor, an antibody, or an aptamer. Continuous purificationof biological products (e.g., a monoclonal antibody) with affinitymagnetic resin beads can avoid the cumbersome processing steps oftraditional affinity column chromatography (e.g., Protein A affinitychromatography).

In embodiments, the magnetic resin beads of the affinity-based, magneticpurification apparatus have a diameter of about 0.2 micron to about 200micron. The diameter of the magnetic resin beads may depend on thebiological product being purified and the flow rate of the process.Moreover, the magnetic resin beads may have a concentration ranging from0.01% to 25% by weight. For example, the concentration of the magneticresin beads may be about 1% to about 10% by weight. In other examples,the binding capacity of the magnetic resin beads is a function of thebead concentration, surface area-to-volume ratio, affinity liganddensity, or any combination thereof. In yet other examples, the magneticresin beads may be solid, porous, nanoporous, microporous, or anycombination thereof.

The loop conveyer system may refer to, for example, a continuous orendless loop. The loop conveyer system is advantageous in that it allowsfor large volumes to move at high flow rates continuously andefficiently through the process, while affording a smaller footprintwhen compared to traditional affinity column chromatography systems(e.g., Protein A affinity chromatography). The biological products areconveyed directly on a track, so both regular and irregular shapedobjects of all sizes can be configured for transport. In some aspects,the object is a transport vessel having a regular shape (e.g., a cube, arectangular prism, a cylinder, a cone).

In embodiments, the loop conveyor system has at least two transportvessels charged with magnetic resin beads that are configured tocontinuously receive a filtrate comprising a mixture containing abiological product and subsequently transport the resultingheterogeneous mixture containing a biological product, magnetic resinbeads, a buffer, or any combination thereof.

The pick and place robotics system may refer to, for example, at leastone robot or robotic arm. The pick and place robotics system isadvantageous in that it allows for large volumes to move at high flowrates continuously and efficiently through the process, while affordinga smaller footprint when compared to traditional affinity columnchromatography systems (e.g., Protein A affinity chromatography). Thebiological products contained in transport vessels are picked andplaced, so regular shaped objects of all sizes can be configured fortransport and stacking, with or without a handle. In some aspects, theobject is a transport vessel having a regular shape (e.g., a cube, arectangular prism).

In embodiments, the pick and place robotics system has at least twotransport vessels charged with magnetic resin beads that are configuredto continuously receive a filtrate comprising a mixture containing abiological product and subsequently transport the resultingheterogeneous mixture containing a biological product, magnetic resinbeads, a buffer, or any combination thereof.

The affinity-based, magnetic purification module further includes atleast one external magnetic field that may be used to attract, and thusseparate, said magnetic resin beads from the heterogeneous mixture toenable washing within at least one of the at least two transportvessels. Further, the at least one external magnetic field may be usedto attract, and thus separate, said magnetic resin beads from theheterogeneous mixture to enable elution of said biological productwithin at least one of the at least two transport vessels.Alternatively, the at least one external magnetic field may be used toenable recycling of said magnetic resin beads within at least one of theat least two transport vessels. In examples, mixing of the magneticresin beads may be accomplished by placing the at least one transportvessel between two separate and opposing magnetic fields that togglebetween states of on and off.

The process described herein also includes continuously transferring thefraction containing the biological product from the at least one firstoutlet of the first module to a second module having at least one inletfor receiving flow from the at least one first outlet of the firstmodule, and the second module comprises a charge-based, magneticpurification apparatus. The “charge-based, magnetic purificationapparatus” as used herein includes, for example, purifying biologicalmolecules based on their surface charge, ionic character, electrostaticinteractions, or isoelectric point. As described herein, thecharge-based, magnetic purification comprises a positive charge-based,magnetic purification apparatus, a negative charge-based, magneticpurification apparatus, or combinations thereof. In examples, the secondmodule has at least one second inlet and at least one second outlet andis configured to permit continuous fluid flow between the second inletand the second outlet via a loop conveyor system or a pick and placerobotics system.

In embodiments, the charge-based, magnetic purification apparatus (e.g.,positive and/or negative charge-based, magnetic purification) furtherincludes a suspension of magnetic resin beads. The surface of themagnetic resin beads, for example, may comprise cationic or anionicfunctionality configured to selectively associate with said biologicalproduct at a specific pH and ionic strength to enable positivecharge-based, magnetic purification or negative charge-based, magneticpurification, respectively. Continuous purification of biologicalproducts (e.g., a monoclonal antibody) with ionic magnetic resin beadscan avoid the cumbersome processing steps of traditional ion-exchangecolumn chromatographies (e.g., cation exchange or anion exchangechromatographies).

In embodiments, the magnetic resin beads of the charge-based, magneticpurification apparatus have a diameter of about 0.2 micron to about 200micron. The diameter of the magnetic resin beads may depend on thebiological product being purified and the flow rate of the process.Moreover, the magnetic resin beads may have a concentration ranging from0.01% to 25% by weight. For example, the concentration of the magneticresin beads may be about 1% to about 10% by weight. In other examples,the charge or electrostatic association capacity of the magnetic resinbeads is a function of the bead concentration, surface area-to-volumeratio, surface charge density, net charge, or any combination thereof.In yet other examples, the magnetic resin beads may be solid, porous,nanoporous, microporous, or any combination thereof.

The loop conveyer system may refer to, for example, a continuous orendless loop. The loop conveyer system is advantageous in that it allowsfor large volumes to move at high flow rates continuously andefficiently through the process, while affording a smaller footprintwhen compared to traditional ion-exchange column chromatography systems.The biological products are conveyed directly on a track, so bothregular and irregular shaped objects of all sizes can be configured fortransport. In some aspects, the object is a transport vessel having aregular shape (e.g., a cube, a rectangular prism, a cylinder, a cone).

In embodiments, the loop conveyor system has at least two transportvessels charged with magnetic resin beads that are configured tocontinuously receive a mixture containing a biological product andsubsequently transport the resulting heterogeneous mixture containing abiological product, magnetic resin beads, a buffer, or any combinationthereof.

The pick and place robotics system may refer to, for example, at leastone robot or robotic arm. The pick and place robotics system isadvantageous in that it allows for large volumes to move at high flowrates continuously and efficiently through the process, while affordinga smaller footprint when compared to traditional ion-exchangechromatography systems. The biological products contained in transportvessels are picked and placed, so regular shaped objects of all sizescan be configured for transport and stacking, with or without a handle.In some aspects, the object is a transport vessel having a regular shape(e.g., a cube, a rectangular prism).

In embodiments, the pick and place robotics system has at least twotransport vessels charged with magnetic resin beads that are configuredto continuously receive a filtrate comprising a mixture containing abiological product and subsequently transport the resultingheterogeneous mixture containing a biological product, magnetic resinbeads, a buffer, or any combination thereof.

The charge-based, magnetic purification module further includes at leastone external magnetic field that may be used to attract, and thusseparate, said magnetic resin beads from the heterogeneous mixture toenable washing within at least one of the at least two transportvessels. Further, the at least one external magnetic field may be usedto attract, and thus separate, said magnetic resin beads from theheterogeneous mixture to enable dissociation and collection of saidbiological product within at least one of the at least two transportvessels. Alternatively, the at least one external magnetic field may beused to enable recycling of said magnetic resin beads within at leastone of the at least two transport vessels. In examples, mixing of themagnetic resin beads may be accomplished by placing the at least onetransport vessel between two separate and opposing magnetic fields thattoggle between states of on and off.

In embodiments described herein, the magnetic resin beads of one or bothof the first (affinity-based, magnetic purification) and/or second(charged-based, magnetic purification) module(s) are recycled andre-used. For example, the beads may be re-used at least 2, 3, 4, or moretimes for purifying a biological product.

Alternatively, the process described herein includes continuouslytransferring the fraction containing the biological product from the atleast one first outlet of the first module to a second module having atleast one inlet for receiving flow from the at least one first outlet ofthe first module, and the second module comprises a free-flowelectrophoresis apparatus. The free-flow electrophoresis apparatushaving a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and an aqueous ionic solution, may be used in lieu of or inaddition to the charge-based, magnetic purification module(s) to purifythe biological product (e.g., a monoclonal antibody).

In examples, the solution contacting surfaces of the two parallel platescomprise glass, ceramic, plastic, or any combination thereof. In someexamples, the aqueous ionic solution may give rise to a pH gradientacross the main separation channel. In other examples, the aqueous ionicsolution may confer constant pH across the main separation channel.

In embodiments, the free-flow electrophoresis apparatus has at least onefluidic device comprising a fluidic channel created between two parallelplates, an electric field or electric field gradient orthogonal to thefluid flow direction, and a pH gradient. In examples, the isoelectricpoint-based, fluidic purification module includes at least one firstfluidic device comprising a fluidic channel created between two parallelplates, an electric field or electric field gradient orthogonal to thefluid flow direction, and a coarse pH gradient across the mainseparation channel (in examples, a coarse pH gradient may be a pH rangefrom about 2 to about 10); and at least one second fluidic devicecomprising a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and a fine pH gradient across the main separation channel (inexamples, a fine pH gradient may be a pH range from about 5 to about 8).In examples, additional, subsequent fluidic devices or chips comprisinga fluidic channel created between two parallel plates and an electricfield or electric field gradient orthogonal to the fluid flow directionmay be used to enable further refining of the pH gradient across themain separation channel (e.g., a pH range from about 7.1 to about 7.6).

In other embodiments, the free-flow electrophoresis apparatus has atleast one fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and no pH gradient (e.g. aconstant pH across the main separation channel) to operate in a zoneelectrophoresis or charge separating mode of operation. In examples, theisoelectric point-based, fluidic purification module includes at leastone first fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and constant basic pH (e.g., apH of greater than 7); and at least one second fluidic device comprisinga fluidic channel created between two parallel plates, an electric fieldor electric field gradient orthogonal to the fluid flow direction, and aconstant acidic pH (e.g., a pH of less than 7).

In other embodiments, the free-flow electrophoresis apparatus has atleast one fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and both an acidic pH gradientand a basic pH gradient separated by a spacer solution (e.g. NaClsolution) to operate in an isotachophoresis mode of operation.

In other embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first free-flow electrophoresis apparatuscomprising a fluidic channel created between two parallel plates and anelectric field or electric field gradient orthogonal to the fluid flowdirection, and at least one second free-flow electrophoresis apparatuscomprising a fluidic channel created between two parallel plates and anelectric field or electric field gradient orthogonal to the fluid flowdirection, wherein each device connected in series and is capable ofoperating in an independent mode of operation to enable purification.For example, the at least one first free-flow electrophoresis apparatusmay operate in an isoelectric focusing mode and the at least one secondfree-flow electrophoresis apparatus may operate in an isotachophoresismode to increase separation resolution.

In other embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first fluidic device comprising fluidicchannel having at least one dielectrophoretic electrode capable ofinducing a defined, unidirectional force; at least one second free-flowelectrophoresis apparatus comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and a coarse pH gradient acrossthe main separation channel (e.g., a pH range from about 2 to about 10);and at least one third free-flow electrophoresis apparatus comprising afluidic channel created between two parallel plates, an electric fieldor electric field gradient orthogonal to the fluid flow direction, and afine pH gradient across the main separation channel (e.g., a pH rangefrom about 5 to about 8). In examples, additional, subsequent fluidicdevices or chips comprising a fluidic channel created between twoparallel plates and an electric field or electric field gradientorthogonal to the fluid flow direction may be used to enable furtherrefining of the pH gradient across the main separation channel (e.g., apH range from about 7.1 to about 7.6).

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least two electrodes (e.g. platinum wireelectrodes) to function as an anode or a cathode.

In embodiments, the backpressure within the isoelectric point-basedfluidic purification apparatus is dependent on the channel geometry anddimensions, the inlet and outlet opening and/or tubing diameters, andthe input flow rate. In examples, the backpressure ranges from about 0.5psi to about 10 psi. In some examples, the backpressure is controlledby, for example, without intent to be limiting, a needle valve.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least one de-bubbler system tocontinuously remove O₂ and H₂ gas bubbles that evolve in the electrodechannels under applied voltage. In some embodiments, removal ofelectrolysis bubbles is essential to enable continuous operation forsubstantially long periods of time. In examples, the de-bubbler systemutilizes a hydrophobic PTFE membrane to create a water-tight seal atopthe electrode channel that permits continuous removal of electrolysisbubbles at the point of generation by exposure to a vacuum system. Inexamples, the vacuum gauge pressure ranges from about −0.05 bar to about−0.4 bar.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises an active cooling system or heat sink (e.g.,a Peltier device, a thermal chuck with a circulating water/propyleneglycol jacket) to enable temperature control and Joule heat dissipation.For example, the active cooling system may control cooling and/or heatdissipation in the range from about 4° C. to about 50° C., preferablyfrom about 4° C. to about 37° C. Ideally, when isolating a biologicalproduct (e.g., a monoclonal antibody), the temperature is maintained atabout 10° C. to about 25° C. In examples, the active cooling systemcomprises an aluminum thermal chuck containing a chilled, circulatingwater/propylene glycol jacket.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one buffer or ampholyte system.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one electrode solution. In some embodiments, the atleast one electrode solution comprises an electrolyte solutionconfigured to contact and enable the appropriate function of an anode ora cathode, for example, phosphoric acid and sodium hydroxide,respectively. In other embodiments, the at least one electrode solutioncomprises at least one ampholyte solution configured to contact andenable the appropriate function of an anode or a cathode, for example,Tris buffered saline flowing through the main separation channel, theanode channel, and the cathode channel.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one sensor or detector. In examples, the at least onesensor or detector is positioned in-line. In some examples, the at leastone sensor or detector includes, but is not limited to, a flow sensor, atemperature sensor, a conductivity sensor, a pH sensor, a refractiveindex detector, a UV detector, or a backpressure sensor.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one liquid circuit breaker or disconnect downstream ofthe device and upstream of the at least one in-line sensor or detectorto ensure the ability to perform sensing or detection in a voltage-freesolution.

The presently claimed process provides for a number of advantages overcurrent downstream methods and processes for purifying a biologicalproduct, for example, a protein or fragment thereof (a polypeptide), anantibody or fragment thereof, a cytokine, a chemokine, an enzyme, or agrowth factor. For example, without intent to be limiting, the processdescribed herein provides a continuous bioprocess for purifying amonoclonal antibody that maintains throughput and yield, whilesignificantly decreasing the production facility footprint, the timerequired for facility buildout and validation, the costs associated withfacility buildout, and capital equipment expenditure, when compared tothe traditional approaches of batch, single-use, or semi-continuousmonoclonal antibody manufacturing. The continuous bioprocessing asdescribed herein affords smaller, more streamlined equipment (e.g.,smaller bioreactor volumes and downstream bioprocess equipment) becausethe ability to operate continuously eliminates the need for the largeprocess equipment required for the batch centrifugation, depthfiltration, and column chromatography steps of traditional downstreambioprocessing, whose size is dictated by large bioreactor volumes.Further, the smaller, more streamlined equipment operating continuouslyaffords the use of significantly smaller bioreactor(s) that producemonoclonal antibodies at steady-state. The continuous bioprocess asdescribed herein may also significantly decrease operating expenditures,overall bioprocess line downtime, and biological product loss whencompared to traditional monoclonal antibody manufacturing approaches.Finally, the process described herein for purifying a biological productis conducted in a system with a footprint that occupies significantlyless square footage than current techniques, without sacrificing productthroughput or yield on a kilograms/year basis.

Advantages of the process and methods described herein include theability to remove large impurities (e.g., cells, cell debris, andaggregates) without membrane fouling or occlusion. Membrane fouling mayrefer to a process whereby the heterogeneous mixture is deposited on themembrane surface or in the membrane pores so that the membrane'sperformance is decreased over time, and thus creating a major limitationin the utility of traditional filtration systems. For example, it isknown in the art that clarification of cells, cell debris and aggregatesfrom cell culture media with traditional filtration or tangential flowfiltration systems typically leads to fouling or occlusion of the filtermembrane, thus rendering these methodologies unsuitable as a means tocontinuously remove large impurities from a heterogeneous mixturecontaining a biological product over long-term continuous processing. Incontrast, the dynamic filtration apparatus described herein enablescontinuous removal of large impurities from a heterogeneous mixturecontaining a biological product without membrane fouling, as the activetarget region of the filter membrane is constantly being refreshed.

Additionally, because the entire process of producing and purifying thebiological product may be continuous and can maintain a flow rate thatranges from about 0.1 mL/minute to about 50 mL/minute across theentirety of the process (e.g., about 5 mL/minute to about 10 mL/minute),the process equipment and overall process footprint is able to have asignificantly smaller footprint than current standard processes, withoutsacrificing product throughput or yield on a kilogram/year basis. Forexample, the process for producing and purifying a monoclonal antibodyas described herein is operated with a footprint that occupies up toabout 30,000 square feet. In contrast, current monoclonal antibodyproduction and downstream processes require at least 200,000 squarefeet. In examples, the process of purifying the biological product has aflow rate that ranges from about 1 mL/minute to about 10 mL/minute. Insome examples, the flow rate of the step of continuously removing largeimpurities from the heterogeneous mixture ranges from about 0.1mL/minute to about 50 mL/minute. In other examples, the flow rate of thestep of continuously removing large impurities from the heterogeneousmixture is equivalent to the flow rate from the bioreactor bleed line.In other examples, the process provides that the flow rate of the stepof continuously transferring the filtrate to a first module ranges fromabout 0.1 mL/minute to about 50 mL/minute. In yet other examples, theprocess provides that the flow rate of the step of continuouslytransferring the fraction containing the biological product from thefirst outlet to a second module ranges from about 0.1 mL/minute to about50 mL/minute.

An important advantage of the process and methods utilizing magneticresin beads (e.g. magnetic agarose) described herein includes that thesesystems do not require traditional stationary phase or packed resincolumns (e.g., for standard chromatographies) to be sanitized, recycledand/or regenerated. For example, these systems provide for recyclingand/or regeneration of the magnetic resin beads to create a limitlesssurface area of the magnetic resin beads during operation, and in turnprovides a continuous and cost-effective method. Put in another way, themodules described herein do not have a fixed binding or associationcapacity. In specific examples, the magnetic resin beads used duringpurification of the biological product, as described herein, areconstantly being recycled and regenerated, and therefore able to acceptflow from the previous step, either a dynamic filtration module or apurification module, without interruption of the flow from thebioreactor bleed line.

Put another way, the modules described in the present invention do nothave to be left idle in order to be sanitized, regenerated and/orrecycled after running, as they are continuously undergoing these steps.The method differs from current continuous chromatographic methods, inthat current column chromatography methods have defined column capacitylimitations due to resin packing constraints and thus require columnswitching of multiple packed columns to accept continuous input flow andenable regeneration and/or recycling of the columns that have reachedfull capacity. Another advantage of the methods described hereinincludes that the magnetic resin beads are not packed into a stationaryphase, rather the magnetic resin beads have mobility. This increases thesurface area of the resin beads that is available for binding orassociation, as substantially more of the magnetic resin bead surface isexposed and free to bind. Additionally, the resin beads in a packedcolumn are exposed to a high pressure differential in order to generateflow through the column and damage from which is one of the reasons forless than desired column lifetime. The mobile resin beads in thepresently described invention are subjected to substantially lowerpressures which is much gentler on the fragile beads, resulting inlonger lifetimes. Additionally, this mobility makes the magnetic resinbeads more likely to be completed regenerated and returned to theirinitial condition. This further adds to the cost-effectiveness of themethods described herein, as the magnetic resin is utilized moreefficiently.

An important advantage of the process and methods utilizing free-flowelectrophoresis described herein includes that this system represents a“no product loss” process, in that, there is no need for the product tointeract with a resin or other purifying moieties, as the separationoccurs in aqueous solution according to the physicochemical propertiesof the target biological product via interaction with an electric field.Another advantage is observed in the resolving power of this approach,as a theoretically higher purity product is achievable when compared totraditional ion-exchange chromatographies. Additionally, the separationbased on intrinsic physicochemical properties extends the utility ofthis approach for the purification a plethora of biological products,including, but not limited to, a protein or fragment thereof (apolypeptide), an antibody or fragment thereof, a cytokine, a chemokine,an enzyme, a growth factor, an oligonucleotide, a virus, an adenovirus,an adeno-associated virus (AAV), or a lentivirus.

Further, the modular approach affords flexibility in process design toaccommodate a diverse range of biological products.

Continuous Process for Purifying a Biological Product Using a DynamicFiltration Module, an Affinity-Based Purification Module, and at LeastOne of a Charge-Based Purification Module or an Isoelectric Point-Based,Fluidic Purification Module.

A continuous process for purifying a biological product is described;the process including continuously receiving, via an input line, aheterogeneous mixture containing the biological product, wherein thebiological product includes, but is not limited to, a protein orfragment thereof (a polypeptide), an antibody or fragment thereof, acytokine, a chemokine, an enzyme, or a growth factor. When purifying,the biological product (e.g., a monoclonal antibody) is substantiallypure when it is at least about 60%, 70%, 80%, 90%, 95%, or even 99%, byweight, free from impurities (cells, cellular debris, aggregates, hostcell proteins, undesired proteins and peptides, undesired antibodies,undesired nucleic acids and oligonucleotides, viruses, salts, buffercomponents, surfactants, sugars, metallic contaminants, leachables,media components, and/or naturally-occurring organic molecules withwhich it is naturally associated).

The process includes continuously removing large impurities from theheterogeneous mixture by dynamic filtration. Said dynamic filtrationprocess includes at least one dynamic filtration module thatcontinuously feeds the biological product from at least one output headin fluid communication with the input line to the dynamic filtrationmodule under negative pressure, thereby producing a filtrate comprisingthe biological product. The dynamic filtration module may furtherinclude at least one additional input line to supply a wash buffer via acoaxial output head or a separate monoaxial output head.

In embodiments, the process described herein includes purifying abiological product that is continuously produced in a bioreactor (e.g.,a fed-batch bioreactor, a perfusion bioreactor, a chemostat bioreactor).For example, the bioreactor includes a bioreactor feed line and anoutput bleed line to enable steady-state cell culture growth conditions,and the output bleed line functions as the input line to permitcontinuous fluid flow from the bioreactor to the dynamic filtrationmodule.

As described herein, the process of continuously removing largeimpurities from the heterogeneous mixture does not includecentrifugation, disk-stack centrifugation, depth filtration, staticfiltration, tangential flow filtration, a hydrocyclone, or anycombination thereof. The term “static filtration” refers to a process inwhich the heterogeneous mixture being filtered remains static, meaning,for example, that the filter membrane (or depth filter) has a definedcapacity, and the rate of filtration decreases as the membrane reachesits capacity (e.g., membrane pores become occluded). In a “static” (asopposed to “dynamic”) filtration, the filter membrane remains stationary(does not move), and the flow (e.g., of the heterogeneous mixture)passes through the stationary filter membrane. These static filtrationmethods are common in the art and are simple and well-understood.

Unlike the static filtration methods commonly used in the art, theprocess herein describes a dynamic filtration module, wherein componentsof the dynamic filtration module move in a coordinated fashion (e.g.,the membrane moves or advances in accordance with the flow rate of theentire process) to enable filtration to occur continuously across afresh, unused target region of filter membrane. This eliminates membranefouling or occlusion and permits control over the filter cake packingand thickness during operation.

The dynamic filtration module includes a filter membrane roll, amembrane support structure, at least one support rod or roller, a vacuumline, a vacuum system, and at least one vacuum collection vessel.

For example, the filter membrane roll includes a rolled filter membrane,wherein the filter membrane, without intent to be limiting, comprisespolyethersulfone (PES), hydrophilic polysulfone, cellulose ester,cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF,polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE,or any combination thereof.

The pore size of the rolled filter membrane depends on the biologicalproduct being purified. In examples, the rolled filter membrane has apore size in the range from 0.1 μm to 1 μm. Alternatively, the pore sizeis in the range from about 0.2 μm to about 0.45 μm, or the pore size isless than about 0.45 μm. In other examples, when purifying an antibody,the pore size of the rolled filter membrane is in the range of 0.2 μm toabout 0.45μm.

The filter membrane roll has a width from about 10 mm to about 600 mm.The width of the filter membrane roll, for example, may depend on thesize of the dynamic filtration system or the membrane support structure.

In embodiments, the filter membrane roll further functions as a feedreel that communicates with a collection reel, meaning the filtermembrane originates from pre-fabricated roll and spans to an initiallyempty collecting roll, thus creating a reel-to-reel system. In aspects,the dynamic filtration module includes a rolled filter membraneextending between a feed reel and a collection reel, the filter membranehaving an active target region that is configured to receive theheterogeneous mixture. In examples, the feed reel motion is governed bya Servo motor coupled with a gear box to limit rotations per minute(RPM) by a ratio of 200:1 to enable low membrane transport velocitieswith high torque. The collection reel motion is governed by a Servomotor coupled with a gear box to limit RPM by a ratio of 200:1 to enablelow membrane transport velocities with high torque. Further, the feedreel motor and the collection reel motor are controlled by a closed-loopcontroller that operates a feedback mechanism to ensure consistentvelocity with the constantly changing diameters of the filter membraneroll on both the feed reel and the collection reel during operation. Inexamples, the feed reel and the collection reel operate in the samedirection with equivalent velocities.

In embodiments, the transport velocity of the filter membrane rangesfrom about 0.1 mm/sec to about 100 mm/sec, preferably from about 0.1mm/sec to about 10 mm/sec.

The membrane support structure of the dynamic filtration module includesa mechanically smooth contact surface derived from a material having alow static coefficient of friction (e.g. PTFE) and an opening that hascontinuity with the vacuum line. As used herein, the “membrane supportstructure” refers to a fabricated component that provides structuralsupport to the active region of the filter membrane, to preventdeformation, as it traverses an area of negative pressure, resultingfrom the opening having continuity with the vacuum line. Further, asused herein, “mechanically smooth contact surface” refers to a surfacehaving a low static coefficient of friction, thus creating a lowfrictional force opposing transport of the filter membrane, especiallywhen wetted. The mechanically smooth contact surface may influence theease at which the filter membrane moves in a dynamic fashion. Themechanically smooth contact surface may also be measured in surfaceroughness, where the lower the value the smoother the surface. Moreover,since rougher surfaces have more friction between them than smoothersurfaces, the mechanically smooth contact surface, as used herein,refers to a surface having lower friction (i.e., a low staticcoefficient of friction).

In embodiments, the membrane support structure of the dynamic filtrationmodule includes an opening. The opening for example, may include a mesh,at least one slot, at least one hole, a frit, a porous material, or anycombination thereof. For example, the opening may include a series ofregularly or irregularly spaced elements (e.g., a mesh, at least oneslot, at least one hole, or any combination thereof). Moreover, theopening may include regularly spaced elements, for example the openingmay include a series of equally spaced, parallel slots. Additionally,the opening can include one grate (e.g., a series of regularly orirregularly spaced elements as described above). In other examples, theopening can include more than one grate, with each grate perpendicular.The opening can be a collection of irregular or regular elements (e.g.,a series of parallel slots). The opening can also include a mesh, whichare of split-thickness or of full-thickness and may or may not be inparallel rows. The elements of the opening (e.g., a mesh, at least oneslot, at least one hole, a frit, a porous material, or any combinationthereof) may be of any desired thickness. For example, without intent tobe limiting, the opening may include a mesh with a thickness of about0.25 mm to about 5 mm.

The membrane support structure of the dynamic filtration module includesa temperature control mechanism. The temperature control mechanismmaintains a temperature from about 4° C. to about 37° C. in the presenceof evaporative cooling. For example, during purification of an antibody,the temperature control mechanism maintains a temperature from about 15°C. to about 37° C. Exemplary temperature control mechanisms include, butare not limited to, single loop controllers, multi-loop controllers,closed loop controllers, PID controllers, Peltier devices, and/orthermal chucks with circulating water/propylene glycol jackets.

In embodiments, the at least one support rod or roller of the dynamicfiltration module has a mechanically smooth contact surface derived froma material having a low static coefficient of friction (e.g. PTFE, PFA).In examples, the dynamic filtration module includes at least one supportrod or roller with a mechanically smooth contact surface to stabilizethe motion of the filter membrane across the membrane support structure.

In embodiments, the dynamic filtration module includes at least oneoutput head for modulating flow of the heterogeneous mixture anddispensing the heterogeneous mixture onto the active target region ofthe filter membrane. In examples, the at least one output head is a tubeor a slot die.

In some embodiments, the dynamic filtration module further includes atleast one additional input line to supply a wash buffer via a coaxialoutput head, a separate monoaxial output head, a separate slot dieoutput head, or a slot die output head with multiple openings.

In some embodiments, the dynamic filtration module includes elementsknown in the coating and converting industry, for example, withoutintent to be limiting, active or passive edge guides, tension control(e.g. a dancer), break and tension detectors, or any combinationthereof.

In embodiments, the dynamic filtration module includes a vacuum systemhaving continuity with the membrane support structure to apply negativepressure across the target region (e.g., active target region) of thefilter membrane, where the negative pressure allows for active transportof the filter membrane across the membrane support structure and enablescollection of the filtrate containing the biological product. Inexamples, the vacuum system of the dynamic filtration module maintains agauge pressure of about −0.05 bar to about −0.98 bar for continuousfiltration.

In embodiments, the dynamic filtration module further includes at leastone vacuum collection vessel configured to collect the filtrate, and atleast one sensor or detector. In aspects described herein, during thepurification by dynamic filtration, the filtrate comprising thebiological product is fed under negative pressure into a vacuumcollection vessel capable of collecting from about 50 mL to about 100 L.In examples, the vacuum collection vessel capable of collecting thefiltrate is from about 1 L to about 10 L. In other examples, the vacuumcollection vessel capable of collecting the filtrate is from about 1 Lto about 50 L.

In embodiments, the process of continuously removing large impurities(e.g., cells, cell debris, and aggregates) from the heterogeneousmixture by dynamic filtration comprises a multiple stage filtration withat least two discrete rolled filter membranes with different pore sizes.In examples, this multiple stage dynamic filtration process includes atleast one first dynamic filtration apparatus having a rolled filtermembrane with a large pore size (e.g., 0.45 μm) in fluid communicationwith at least one second dynamic filtration apparatus having a rolledfilter membrane with a small pore size (e.g., 0.2 μm), thereby producinga filtrate comprising the biological product.

The process described herein includes continuously transferring thefiltrate to a first module capable of separating the solution into twoor more fractions comprising at least one fraction containing thebiological product. For example, separating the solution into two ormore fractions, may include at least one fraction containing thebiological product, and the at least one other fraction containing smallimpurities. As described herein, the first module comprises anaffinity-based purification apparatus. The “affinity-based purificationapparatus” refers to a purification technique based upon molecular,conformational binding interactions (e.g., ligand-receptor interactions)in which selective surface-immobilized ligands recognize and bind to thebiological product to be purified. In examples, the first module has atleast one first inlet and at least one first outlet and is configured topermit continuous fluid flow between the first inlet and the firstoutlet via a mechanical rotary system comprising a lid system, a vesselcarousel, and a collection system.

In embodiments, the affinity-based purification apparatus furtherincludes a suspension of resin beads. The surface of the resin beads,for example, without intent to be limiting, is coupled with Protein A,Protein G, Protein L, an antigenic protein, a protein, a receptor, anantibody, or an aptamer. Continuous purification of biological products(e.g., a monoclonal antibody) with affinity resin beads can avoid thecumbersome processing steps of traditional affinity columnchromatography (e.g., Protein A affinity chromatography).

In embodiments, the resin beads of the affinity-based purificationapparatus have a diameter of about 0.2 micron to about 200 micron. Thediameter of the resin beads may depend on the biological product beingpurified and the flow rate of the process. Moreover, the resin beads mayhave a concentration ranging from 0.01% to 25% by weight. For example,the concentration of the magnetic resin beads may be about 1% to about20% by weight. In other examples, the binding capacity of the resinbeads is a function of the bead concentration, surface area-to-volumeratio, affinity ligand density, or any combination thereof. In yet otherexamples, the resin beads may be solid, porous, nanoporous, microporous,or any combination thereof.

In embodiments, the affinity-based purification module includes lidsystem having at least one gasketed lid, the at least one gasketed lidcomprising at least one inlet to introduce a gas to enable control ofpositive head pressure, at least one vent port to enable equilibrationto atmospheric pressure, at least one inlet to introduce a suspension ofresin beads; at least one inlet to receive the filtrate containing abiological product, at least two inlets to introduce a buffer system todisperse the resin beads to enable washing of, elution from, orregeneration of said resin beads. In some embodiments, the at least onegasketed lid further comprises a port to accept an overhead stirringimpeller to enable dispersion of the resin beads. In examples, the lidsystem has control of motion along the z-axis.

In embodiments, the affinity-based purification module includes amechanical rotary system, for example, a carousel comprising at leasttwo vessels charged with resin beads that are configured to continuouslyreceive a mixture containing a biological product and subsequentlytransport the resulting heterogeneous mixture containing a biologicalproduct, resin beads, a buffer, or any combination thereof. In examples,the mechanical rotary system is configured to mate with the lid systemto enable pressurization. In other examples, the mechanical rotarysystem has control of motion or rotation in the xy-plane.

In embodiments, the at least two vessels of the affinity-basedpurification module each have a supported, basement filter or filtermembrane to enable retention of the resin beads during process steps ofbinding, de-binding, washing, elution, and regeneration. In examples,the at least two vessels further include a valve to control liquid flow.

In embodiments, the affinity-based purification module includes acollection system that interfaces with at least one of the at least twovessels of the mechanical rotary system to enable collection of waste,the fraction containing the biological product, or any combinationthereof. In examples, the collection system has control of motion alongthe z-axis.

The process described herein also includes continuously transferring thefraction containing the biological product from the at least one firstoutlet of the first module to a second module having at least one inletfor receiving flow from the at least one first outlet of the firstmodule, and the second module comprises a charge-based purificationapparatus. The “charge-based purification apparatus” as used hereinincludes, for example, purifying biological molecules based on theirsurface charge, ionic character, electrostatic interactions, orisoelectric point. As described herein, the charge-based purificationcomprises a positive charge-based purification apparatus, anegative-charge based purification apparatus, or combinations thereof.In examples, the second module has at least one second inlet and atleast one second outlet and is configured to permit continuous fluidflow between the second inlet and the second outlet via a mechanicalrotary system comprising a lid system, a vessel carousel, and acollection system.

In embodiments, the charge-based purification apparatus (e.g., positiveand/or negative charge-based purification) further includes a suspensionof resin beads. The surface of the resin beads, for example, maycomprise cationic or anionic functionality configured to selectivelyassociate with said biological product at a specific pH and ionicstrength to enable positive charge-based purification or negativecharge-based purification, respectively. Continuous purification ofbiological products (e.g., a monoclonal antibody) with ionic resin beadscan avoid the cumbersome processing steps of traditional ion-exchangecolumn chromatographies (e.g., cation exchange or anion exchangechromatographies).

In embodiments, the resin beads of the charge-based purificationapparatus have a diameter of about 0.2 micron to about 200 micron. Thediameter of the resin beads may depend on the biological product beingpurified and the flow rate of the process. Moreover, the resin beads mayhave a concentration ranging from 0.01% to 25% by weight. For example,the concentration of the magnetic resin beads may be about 1% to about20% by weight. In other examples, the charge or electrostaticassociation capacity of the resin beads is a function of the beadconcentration, surface area-to-volume ratio, surface charge density, netcharge, or any combination thereof. In yet other examples, the resinbeads may be solid, porous, nanoporous, microporous, or any combinationthereof.

In embodiments, the charge-based purification module includes lid systemhaving at least one gasketed lid, the at least one gasketed lidcomprising at least one inlet to introduce a gas to enable control ofpositive head pressure, at least one vent port to enable equilibrationto atmospheric pressure, at least one inlet to introduce a suspension ofresin beads; at least one inlet to receive the filtrate containing abiological product, at least two inlets to introduce a buffer system todisperse the resin beads to enable washing of, dissociation from, orregeneration of said resin beads. In some embodiments, the at least onegasketed lid further comprises a port to accept an overhead stirringimpeller to enable dispersion of the resin beads. In examples, the lidsystem has control of motion along the z-axis.

In embodiments, the charge-based purification module includes amechanical rotary system, for example, a carousel comprising at leasttwo vessels charged with resin beads that are configured to continuouslyreceive a mixture containing a biological product and subsequentlytransport the resulting heterogeneous mixture containing a biologicalproduct, resin beads, a buffer, or any combination thereof. In examples,the mechanical rotary system is configured to mate with the lid systemto enable pressurization. In other examples, the mechanical rotarysystem has control of motion or rotation in the xy-plane.

In embodiments, the at least two vessels of the charge-basedpurification module each have a supported, basement filter or filtermembrane to enable retention of the resin beads during process steps ofassociation, dissociation, washing, and regeneration. In examples, theat least two vessels further include a valve to control liquid flow.

In embodiments, the charge-based purification module includes acollection system that interfaces with at least one of the at least twovessels of the mechanical rotary system to enable collection of waste,the fraction containing the biological product, or any combinationthereof. In examples, the collection system has control of motion alongthe z-axis.

In embodiments described herein, the resin beads of one or both of thefirst (affinity-based purification) and/or second (charged-basedpurification) module(s) are recycled and re-used. For example, saidbeads may be re-used at least 2, 3, 4, or more times for purifying abiological product.

Alternatively, the process described herein includes continuouslytransferring the fraction containing the biological product from the atleast one first outlet of the first module to a second module having atleast one inlet for receiving flow from the at least one first outlet ofthe first module, and the second module comprises a free-flowelectrophoresis apparatus. The free-flow electrophoresis apparatushaving a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and an aqueous ionic solution, may be used in lieu of or inaddition to the charge-based, magnetic purification module(s) to purifythe biological product (e.g., a monoclonal antibody).

In examples, the solution contacting surfaces of the two parallel platescomprise glass, ceramic, plastic, or any combination thereof. In someexamples, the aqueous ionic solution may give rise to a pH gradientacross the main separation channel. In other examples, the aqueous ionicsolution may confer constant pH across the main separation channel.

In embodiments, the free-flow electrophoresis apparatus has at least onefluidic device comprising a fluidic channel created between two parallelplates, an electric field or electric field gradient orthogonal to thefluid flow direction, and a pH gradient. In examples, the isoelectricpoint-based, fluidic purification module includes at least one firstfluidic device comprising a fluidic channel created between two parallelplates, an electric field or electric field gradient orthogonal to thefluid flow direction, and a coarse pH gradient across the mainseparation channel (in examples, a coarse pH gradient may be a pH rangefrom about 2 to about 10); and at least one second fluidic devicecomprising a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and a fine pH gradient across the main separation channel (inexamples, a fine pH gradient may be a pH range from about 5 to about 8).In examples, additional, subsequent fluidic devices or chips comprisinga fluidic channel created between two parallel plates and an electricfield or electric field gradient orthogonal to the fluid flow directionmay be used to enable further refining of the pH gradient across themain separation channel (e.g., a pH range from about 7.1 to about 7.6).

In other embodiments, the free-flow electrophoresis apparatus has atleast one fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and no pH gradient to operate ina zone electrophoresis or charge separating mode of operation. Inexamples, the isoelectric point-based, fluidic purification moduleincludes at least one first fluidic device comprising a fluidic channelcreated between two parallel plates, an electric field or electric fieldgradient orthogonal to the fluid flow direction, and constant basic pH(e.g., a pH of greater than 7); and at least one second fluidic devicecomprising a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and a constant acidic pH (e.g., a pH of less than 7).

In other embodiments, the free-flow electrophoresis apparatus has atleast one fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and both an acidic pH gradientand a basic pH gradient separated by a spacer solution (e.g. NaClsolution) to operate in an isotachophoresis mode of operation.

In other embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first free-flow electrophoresis apparatuscomprising a fluidic channel created between two parallel plates and anelectric field or electric field gradient orthogonal to the fluid flowdirection, and at least one second free-flow electrophoresis apparatuscomprising a fluidic channel created between two parallel plates and anelectric field or electric field gradient orthogonal to the fluid flowdirection, wherein each device connected in series and is capable ofoperating in an independent mode of operation to enable purification.For example, the at least one first free-flow electrophoresis apparatusmay operate in an isoelectric focusing mode and the at least one secondfree-flow electrophoresis apparatus may operate in an isotachophoresismode to increase separation resolution.

In other embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first fluidic device comprising fluidicchannel having at least one dielectrophoretic electrode capable ofinducing a defined, unidirectional force; at least one second free-flowelectrophoresis apparatus comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and a coarse pH gradient acrossthe main separation channel (e.g., a pH range from about 2 to about 10);and at least one third free-flow electrophoresis apparatus comprising afluidic channel created between two parallel plates, an electric fieldor electric field gradient orthogonal to the fluid flow direction, and afine pH gradient across the main separation channel (e.g., a pH rangefrom about 5 to about 8). In examples, additional, subsequent fluidicdevices or chips comprising a fluidic channel created between twoparallel plates and an electric field or electric field gradientorthogonal to the fluid flow direction may be used to enable furtherrefining of the pH gradient across the main separation channel (e.g., apH range from about 7.1 to about 7.6).

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least two electrodes (e.g. platinum wireelectrodes) to function as an anode or a cathode.

In embodiments, the backpressure within the isoelectric point-basedfluidic purification apparatus is dependent on the channel geometry anddimensions, the inlet and outlet opening and/or tubing diameters, andthe input flow rate. In examples, the backpressure ranges from about 0.5psi to about 10 psi. In some examples, the backpressure is controlledby, for example, without intent to be limiting, a needle valve.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least one de-bubbler system tocontinuously remove O₂ and H₂ gas bubbles that evolve in the electrodechannels under applied voltage. In some embodiments, removal ofelectrolysis bubbles is essential to enable continuous operation forsubstantially long periods of time. In examples, the de-bubbler systemutilizes a hydrophobic PTFE membrane to create a water-tight seal atopthe electrode channel that permits continuous removal of electrolysisbubbles at the point of generation by exposure to a vacuum system. Inexamples, the vacuum gauge pressure ranges from about −0.05 bar to about−0.4 bar.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises an active cooling system or heat sink (e.g.,a Peltier device, a thermal chuck with a circulating water/propyleneglycol jacket) to enable temperature control and Joule heat dissipation.For example, the active cooling system may control cooling and/or heatdissipation in the range from about 4° C. to about 50° C., preferablyfrom about 4° C. to about 37° C. Ideally, when isolating a biologicalproduct (e.g., a monoclonal antibody), the temperature is maintained atabout 10° C. to about 25° C. In examples, the active cooling systemcomprises an aluminum thermal chuck containing a chilled, circulatingwater/propylene glycol jacket.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one buffer or ampholyte system.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one electrode solution. In some embodiments, the atleast one electrode solution comprises an electrolyte solutionconfigured to contact and enable the appropriate function of an anode ora cathode, for example, phosphoric acid and sodium hydroxide,respectively. In other embodiments, the at least one electrode solutioncomprises at least one ampholyte solution configured to contact andenable the appropriate function of an anode or a cathode, for example,Tris buffered saline flowing through the main separation channel, theanode channel, and the cathode channel.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one sensor or detector. In examples, the at least onesensor or detector is positioned in-line. In some examples, the at leastone sensor or detector includes, but is not limited to, a flow sensor, atemperature sensor, a conductivity sensor, a pH sensor, a refractiveindex detector, a UV detector, or a backpressure sensor.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one liquid circuit breaker or disconnect downstream ofthe device and upstream of the at least one in-line sensor or detectorto ensure the ability to perform sensing or detection in a voltage-freesolution.

The presently claimed process provides for a number of advantages overcurrent downstream methods and processes for purifying a biologicalproduct, for example, a protein or fragment thereof (a polypeptide), anantibody or fragment thereof, a cytokine, a chemokine, an enzyme, or agrowth factor. For example, without intent to be limiting, the processdescribed herein provides a continuous bioprocess for purifying amonoclonal antibody that maintains throughput and yield, whilesignificantly decreasing the production facility footprint, the timerequired for facility buildout and validation, the costs associated withfacility buildout, and capital equipment expenditure, when compared tothe traditional approaches of batch, single-use, or semi-continuousmonoclonal antibody manufacturing. The continuous bioprocessing asdescribed herein affords smaller, more streamlined equipment (e.g.,smaller bioreactor volumes and downstream bioprocess equipment) becausethe ability to operate continuously eliminates the need for the largeprocess equipment required for the centrifugation, depth filtration, andcolumn chromatography steps of traditional downstream bioprocessing,whose size is dictated by large bioreactor volumes. Further, thesmaller, more streamlined equipment operating continuously affords theuse of significantly smaller bioreactor(s) that produce monoclonalantibodies at steady-state. The continuous bioprocess as describedherein may also significantly decrease operating expenditures, overallbioprocess line downtime, and biological product loss when compared totraditional monoclonal antibody manufacturing approaches. Finally, theprocess described herein for purifying a biological product is conductedin a system with a footprint that occupies significantly less squarefootage than current techniques, without sacrificing product throughputor yield on a kilograms/year basis.

Advantages of the process and methods described herein include theability to remove large impurities (e.g., cells, cell debris, andaggregates) without membrane fouling or occlusion. For example, it isknown in the art that clarification of cells, cell debris and aggregatesfrom cell culture media with traditional filtration or tangential flowfiltration systems typically leads to fouling or occlusion of the filtermembrane, thus rendering these methodologies unsuitable as a means tocontinuously remove large impurities from a heterogeneous mixturecontaining a biological product over long-term continuous processing. Incontrast, the dynamic filtration apparatus described herein enablescontinuous removal of large impurities from a heterogeneous mixturecontaining a biological product without membrane fouling, as the activetarget region of the filter membrane is constantly being refreshed.Additionally, because the entire process of producing and purifying thebiological product may be continuous and can maintain a flow rate thatranges from about 0.1 mL/minute to about 50 mL/minute across theentirety of the process, the process equipment and overall processfootprint is able to have a significantly smaller footprint than currentstandard processes, without sacrificing product throughput or yield on akilogram/year basis. For example, the process for producing andpurifying a monoclonal antibody as described herein is operated with afootprint that occupies up to about 30,000 square feet. In contrast,current monoclonal antibody production and downstream processes requireat least 200,000 square feet. In examples, the process of purifying thebiological product has a flow rate that ranges from about 1 mL/minute toabout 10 mL/minute. In some examples, the flow rate of the step ofcontinuously removing large impurities from the heterogeneous mixtureranges from about 0.1 mL/minute to about 50 mL/minute. In otherexamples, the flow rate of the step of continuously removing largeimpurities from the heterogeneous mixture is equivalent to the flow ratefrom the bioreactor bleed line. In other examples, the process providesthat the flow rate of the step of continuously transferring the filtrateto a first module ranges from about 0.1 mL/minute to about 50 mL/minute.In yet other examples, the process provides that the flow rate of thestep of continuously transferring the fraction containing the biologicalproduct from the first outlet to a second module ranges from about 0.1mL/minute to about 50 mL/minute.

An important advantage of the process and methods utilizing resin beads(e.g. agarose) described herein includes that these systems do notrequire traditional stationary phase or packed resin columns (e.g., forstandard chromatographies) to be sanitized, recycled and/or regenerated.For example, these systems provide for recycling and/or regeneration ofthe resin beads to create a limitless surface area of the resin beadsduring operation, and in turn provides a continuous and cost-effectivemethod. Put in another way, the modules described herein do not have afixed binding or association capacity. In specific examples, the resinbeads used during purification of the biological product, as describedherein, are constantly being recycled and regenerated, and thereforeable to accept flow from the previous step, either a dynamic filtrationmodule or a purification module, without interruption of the flow fromthe bioreactor bleed line. Put another way, the modules described in thepresent invention do not have to be left idle in order to be sanitized,regenerated and/or recycled after running, as they are continuouslyundergoing these steps. The method differs from current continuouschromatographic methods, in that current column chromatography methodshave defined column capacity limitations due to column packingconstraints and thus require column switching of multiple packed columnsto accept continuous input flow and enable regeneration and/or recyclingof the columns that have reached full capacity. Another advantage of themethods described herein includes the that the resin beads are notpacked into a stationary phase, rather the resin beads have mobility.This increases the surface area of the resin beads that is available forbinding or association, as substantially more of the resin bead surfaceis exposed and free to bind. Additionally, the resin beads in a packedcolumn are exposed to a high pressure differential in order to generateflow through the column and damage from which is one of the reasons forless than desired column lifetime. The mobile resin beads in thepresently described invention are subjected to substantially lowerpressures which is much gentler on the fragile beads, resulting inlonger lifetimes. Additionally, this mobility makes the resin beads morelikely to be completed regenerated and returned to their initialcondition. This further adds to the cost-effectiveness of the methodsdescribed herein, as the resin is utilized more efficiently.

An important advantage of the process and methods utilizing free-flowelectrophoresis described herein includes that this system represents a“no product loss” process, in that, there is no need for the product tointeract with a resin or other purifying moieties, as the separationoccurs in aqueous solution according to the physicochemical propertiesof the target biological product via interaction with an electric field.Another advantage is observed in the resolving power of this approach,as a theoretically higher purity product is achievable when compared totraditional ion-exchange chromatographies. Additionally, the separationbased on intrinsic physicochemical properties extends the utility ofthis approach for the purification a plethora of biological products,including, but not limited to, a protein or fragment thereof (apolypeptide), an antibody or fragment thereof, a cytokine, a chemokine,an enzyme, a growth factor, an oligonucleotide, a virus, an adenovirus,an adeno-associated virus (AAV), or a lentivirus.

Further, the modular approach affords flexibility in process design toaccommodate a diverse range of biological products.

Continuous Process for Purifying a Biological Product Using a DynamicFiltration Module, an Affinity-Based, Fluidic Purification Module, andat Least One of a Charge-Based, Fluidic Purification Module or anIsoelectric Point-Based, Fluidic Purification Module

A continuous process for purifying a biological product is described;the process including continuously receiving, via an input line, aheterogeneous mixture containing the biological product, wherein thebiological product includes, but is not limited to, a protein orfragment thereof (a polypeptide), an antibody or fragment thereof, acytokine, a chemokine, an enzyme, or a growth factor. When purifying,the biological product (e.g., a monoclonal antibody) is substantiallypure when it is at least about 60%, 70%, 80%, 90%, 95%, or even 99%, byweight, free from impurities (e.g., cells, cellular debris, aggregates,host cell proteins, undesired proteins and peptides, undesiredantibodies, undesired nucleic acids and oligonucleotides, viruses,salts, buffer components, surfactants, sugars, metallic contaminants,leachables, media components, and/or naturally-occurring organicmolecules with which it is naturally associated).

The process includes continuously removing large impurities from theheterogeneous mixture by dynamic filtration. Said dynamic filtrationprocess includes at least one dynamic filtration module thatcontinuously feeds the biological product from at least one output headin fluid communication with the input line to the dynamic filtrationmodule under negative pressure, thereby producing a filtrate comprisingthe biological product. The dynamic filtration module may furtherinclude at least one additional input line to supply a wash buffer via acoaxial output head or a separate monoaxial output head.

In embodiments, the process described herein includes purifying abiological product that is continuously produced in a bioreactor (e.g.,a fed-batch bioreactor, a perfusion bioreactor, a chemostat bioreactor).For example, the bioreactor includes a bioreactor feed line and anoutput bleed line to enable steady-state cell culture growth conditions,and the output bleed line functions as the input line to permitcontinuous fluid flow from the bioreactor to the dynamic filtrationmodule.

As described herein, the process of continuously removing largeimpurities from the heterogeneous mixture does not includecentrifugation, disk-stack centrifugation, depth filtration, staticfiltration, tangential flow filtration, a hydrocyclone, or anycombination thereof. The term “static filtration” refers to a process inwhich the heterogeneous mixture being filtered remains static, meaningfor example that the filter membrane (or depth filter) has a definedcapacity, and the rate of filtration decreases as the membrane reachesits capacity (e.g., membrane pores become occluded). In a “static” (asopposed to “dynamic”) filtration, the filter membrane remains stationary(does not move), and the flow (e.g., of the heterogeneous mixture)passes through the stationary filter membrane. These static filtrationmethods are common in the art and are simple and well-understood.

Unlike the static filtration methods commonly used in the art, theprocess herein describes a dynamic filtration module, wherein componentsof the dynamic filtration module move in a coordinated fashion (e.g.,the membrane moves or advances in accordance with the flow rate of theentire process) to enable filtration to occur continuously across afresh, unused target region of filter membrane. This eliminates membranefouling or occlusion and permits control over the filter cake packingand thickness during operation.

The dynamic filtration module includes a filter membrane roll, amembrane support structure, at least one support rod or roller, a vacuumline, a vacuum system, and at least one vacuum collection vessel.

In embodiments, the filter membrane roll includes a filter roll, whereinthe filter membrane, without intent to be limiting, comprisespolyethersulfone (PES), hydrophilic polysulfone, cellulose ester,cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF,polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE,or any combination thereof.

The pore size of the rolled filter membrane depends on the biologicalproduct being purified. In examples, the rolled filter membrane has apore size in the range from 0.1 μm to 1 μm. Alternatively, the pore sizeis in the range from about 0.2 μm to about 0.45 μm, or the pore size isless than about 0.45 μm. In other examples, when purifying an antibody,the pore size of the rolled filter membrane is in the range of 0.2 μm toabout 0.45μm.

The filter membrane roll has a width from about 10 mm to about 600 mm.The width of the filter membrane roll, for example, may depend on thesize of the dynamic filtration system or the membrane support structure.

In embodiments, the filter membrane roll further functions as a feedreel that communicates with a collection reel, meaning the filtermembrane originates from pre-fabricated roll and spans to an initiallyempty collecting roll, thus creating a reel-to-reel system. In aspects,the dynamic filtration module includes a rolled filter membraneextending between a feed reel and a collection reel, the filter membranehaving an active target region that is configured to receive theheterogeneous mixture. In examples, the feed reel motion is governed bya Servo motor coupled with a gear box to limit rotations per minute(RPM) by a ratio of 200:1 to enable low membrane transport velocitieswith high torque. The collection reel motion is governed by a Servomotor coupled with a gear box to limit RPM by a ratio of 200:1 to enablelow membrane transport velocities with high torque. Further, the feedreel motor and the collection reel motor are controlled by a closed-loopcontroller that operates a feedback mechanism to ensure consistentvelocity with the constantly changing diameters of the filter membraneroll on both the feed reel and the collection reel during operation. Inexamples, the feed reel and the collection reel operate in the samedirection with equivalent velocities.

In embodiments, the transport velocity of the filter membrane rangesfrom about 0.1 mm/sec to about 100 mm/sec, preferably from about 0.1mm/sec to about 10 mm/sec.

The membrane support structure of the dynamic filtration module includesa mechanically smooth contact surface derived from a material having alow static coefficient of friction (e.g. PTFE) and an opening that hascontinuity with the vacuum line. As used herein, the “membrane supportstructure” refers to a fabricated component that provides structuralsupport to the active region of the filter membrane, to preventdeformation, as it traverses an area of negative pressure, resultingfrom the opening having continuity with the vacuum line. Further, asused herein, “mechanically smooth contact surface” refers to a surfacehaving a low static coefficient of friction, thus creating a lowfrictional force opposing transport of the filter membrane, especiallywhen wetted. The mechanically smooth contact surface may influence theease at which the filter membrane moves in a dynamic fashion. Themechanically smooth contact surface may also be measured in surfaceroughness, where the lower the value the smoother the surface. Moreover,since rougher surfaces have more friction between them than smoothersurfaces, the mechanically smooth contact surface, as used herein,refers to a surface having lower friction (i.e., a low staticcoefficient of friction).

In embodiments, the membrane support structure of the dynamic filtrationmodule includes an opening. The opening for example, may include a mesh,at least one slot, at least one hole, a frit, a porous material, or anycombination thereof. For example, the opening may include a series ofregularly or irregularly spaced elements (e.g., a mesh, at least oneslot, at least one hole, or any combination thereof). Moreover, theopening may include regularly spaced elements, for example the openingmay include a series of equally spaced, parallel slots. Additionally,the opening can include one grate (e.g., a series of regularly orirregularly spaced elements as described above). In other examples, theopening can include more than one grate, with each grate perpendicular.The opening can be a collection of irregular or regular elements (e.g.,a series of parallel slots). The opening can also include a mesh, whichare of split-thickness or of full-thickness and may or may not be inparallel rows. The elements of the opening (e.g., a mesh, at least oneslot, at least one hole, a frit, a porous material, or any combinationthereof) may be of any desired thickness. For example, without intent tobe limiting, the opening may include a mesh with a thickness of about0.25 mm to about 5 mm.

The membrane support structure of the dynamic filtration module includesa temperature control mechanism. The temperature control mechanismmaintains a temperature from about 4° C. to about 37° C. in the presenceof evaporative cooling. For example, during purification of an antibody,the temperature control mechanism maintains a temperature from 15° C. to37° C. Exemplary temperature control mechanisms include, but are notlimited to, single loop controllers, multi-loop controllers, closed loopcontrollers, PID controllers, Peltier devices, resistive heatingelements, and/or thermal chucks with circulating water jackets.

In embodiments, the at least one support rod or roller of the dynamicfiltration module has a mechanically smooth contact surface derived froma material having a low static coefficient of friction (e.g. PTFE, PFA).In examples, the dynamic filtration module includes at least one supportrod or roller with a mechanically smooth contact surface to stabilizethe motion of the filter membrane across the membrane support structure.

In embodiments, the dynamic filtration module includes at least oneoutput head for modulating flow of the heterogeneous mixture anddispensing the heterogeneous mixture onto the target region (e.g.,active target region) of the filter membrane. In examples, the at leastone output head is a tube or a slot die.

In some embodiments, the dynamic filtration module further includes atleast one additional input line to supply a wash buffer via a coaxialoutput head, a separate monoaxial output head, a separate slot dieoutput head, or a slot die output head with multiple openings.

In some embodiments, the dynamic filtration module includes elementsknown in the coating and converting industry, for example, withoutintent to be limiting, active or passive edge guides, tension control(e.g. a dancer), break and tension detectors, or any combinationthereof.

In embodiments, the dynamic filtration module includes a vacuum systemhaving continuity with the membrane support structure to apply negativepressure across the active target region of the filter membrane, wherethe negative pressure allows for active transport of the filter membraneacross the membrane support structure and enables collection of thefiltrate containing the biological product. In examples, the vacuumsystem of the dynamic filtration module maintains a gauge pressure ofabout −0.05 bar to about −0.98 bar for continuous filtration.

In embodiments, the dynamic filtration module further includes at leastone vacuum collection vessel configured to collect the filtrate, and atleast one sensor or detector. In aspects described herein, during thepurification by dynamic filtration, the filtrate comprising thebiological product is fed under negative pressure into a vacuumcollection vessel capable of collecting from about 50 mL to about 100 L.In examples, the vacuum collection vessel capable of collecting thefiltrate is from about 1 L to about 10 L. In other examples, the vacuumcollection vessel capable of collecting the filtrate is from about 1 Lto about 50 L.

In embodiments, the process of continuously removing large impurities(e.g., cells, cell debris, and aggregates) from the heterogeneousmixture by dynamic filtration comprises a multiple stage filtration withat least two discrete rolled filter membranes with different pore sizes.In examples, this multiple stage dynamic filtration process includes atleast one first dynamic filtration apparatus having a rolled filtermembrane with a large pore size (e.g., 0.45 μm) in fluid communicationwith at least one second dynamic filtration apparatus having a rolledfilter membrane with a small pore size (e.g., 0.2 μm), thereby producinga filtrate comprising the biological product.

The process described herein includes continuously transferring thefiltrate to a first module capable of separating the solution into twoor more fractions including at least one fraction containing thebiological product. For example, separating the solution into two ormore fractions, may include at least one fraction containing thebiological product, and the at least one other fraction containing smallimpurities. As described herein, the first module comprises anaffinity-based, fluidic purification apparatus. The “affinity-based,fluidic purification apparatus” refers to a purification technique basedon utilizing molecular, conformational binding interactions (e.g.,ligand-receptor interactions) in which selective surface-immobilizedligands recognize and bind to the biological product to be purified withat least one hybrid fluidic device or chip (e.g., microfluidic,mesofluidic, millifluidic, macrofluidic). In examples, the first modulehas at least one first inlet and at least one first outlet and isconfigured to permit continuous fluid flow between the first inlet andthe first outlet.

In embodiments, the affinity-based, fluidic purification apparatusfurther includes a suspension of magnetic resin beads. The surface ofthe magnetic resin beads, for example, without intent to be limiting, iscoupled with Protein A, Protein G, Protein L, an antigenic protein, aprotein, a receptor, an antibody, or an aptamer. Continuous purificationof biological products (e.g., a monoclonal antibody) with affinitymagnetic resin beads can avoid the cumbersome processing steps oftraditional affinity column chromatography (e.g., Protein A affinitychromatography).

In embodiments, the magnetic resin beads of the affinity-based, fluidicpurification apparatus have a diameter of about 0.2 micron to about 200micron. The diameter of the magnetic resin beads may depend on thebiological product being purified and the flow rate of the process.Moreover, the magnetic resin beads may have a concentration ranging from0.01% to 25% by weight. For example, the concentration of the magneticresin beads may be about 1% by weight. In other examples, the bindingcapacity of the magnetic resin beads is a function of the beadconcentration, surface area-to-volume ratio, affinity ligand density, orany combination thereof. In yet other examples, the magnetic resin beadsmay be solid, porous, nanoporous, microporous, or any combinationthereof.

The at least one hybrid fluidic device or chip of the affinity-based,fluidic purification apparatus may refer to, for example, amicrofluidic, a mesofluidic, a millifluidic, a macrofluidic device orchip, or any combination thereof. In some examples, the fluidic deviceor chip is a hybrid microfluidic device or chip, for example, amicrofluidic device that combines the functionality of cross-flow fluiddynamics with magnetophoretic and dielectrophoretic capabilities,wherein the cross-flow fluid dynamics are governed by the microchanneldesign, the magnetophoresis is accomplished via an external magneticfield, and the dielectrophoresis is accomplished via a dielectrophoreticelectrode. In aspects, the combination of the dielectrophoreticelectrode and the external magnetic field is used to manipulate the flowpath of the magnetic resin beads in the cross-flow microchannel toenable efficient purification at high flow rates (e.g., greater than 0.5mL/min), a phenomenon not currently realized in the field ofmicrofluidics in which flow rates are traditionally limited to μL/hr orμL/min. In other examples, the hybrid fluidic is a microfluidic devicethat combines the functionality of cross-flow fluid dynamics withmagnetophoretic and acoustophoretic capabilities, wherein the cross-flowfluid dynamics are governed by the microchannel design, themagnetophoresis is accomplished via an external magnetic field, and theacoustophoresis is accomplished via a piezoelectric transducer orcrystal. In aspects, the combination of the piezoelectric transducer andthe external magnetic field is used to manipulate the flow path of themagnetic resin beads in the cross-flow microchannel to enable efficientpurification at high flow rates (e.g., greater than 0.5 mL/min), aphenomenon not currently realized in the field of microfluidics in whichflow rates are traditionally limited to μL/hr or μL/min.

In embodiments, the affinity-based, fluidic purification module furtherincludes at least one tangential flow filtration system operated infed-batch or perfusion mode to concentrate and buffer exchange thefraction containing the biological product.

The process as described herein also includes continuously transferringthe fraction containing the biological product from the at least onefirst outlet of the first module to a second module having at least oneinlet for receiving flow from the at least one first outlet of the firstmodule, and the second module comprises a charge-based, fluidicpurification apparatus. The “charge-based, fluidic purificationapparatus” as used herein includes, for example, purifying biologicalmolecules based on their surface charge, ionic character, electrostaticinteractions, or isoelectric point with at least one hybrid fluidicdevice or chip (e.g., a microfluidic, a mesofluidic, a millifluidic, amacrofluidic device or chip, or any combination thereof). As describedherein, the charge-based, fluidic purification comprises a positivecharge-based, fluidic purification apparatus, negative charge-based,fluidic purification apparatus, or combinations thereof. In examples,the second module has at least one second inlet and at least one secondoutlet and is configured to permit continuous fluid flow between thesecond inlet and the second outlet.

In embodiments, the charge-based, fluidic purification apparatus furtherincludes a suspension of magnetic resin beads. The surface of themagnetic resin beads, for example, may comprise cationic or anionicfunctionality configured to selectively associate with said biologicalproduct at a specific pH and ionic strength to enable positivecharge-based, fluidic purification or negative charge-based, fluidicpurification, respectively. Continuous purification of biologicalproducts (e.g., a monoclonal antibody) with ionic magnetic resin beadscan avoid the cumbersome processing steps of traditional ion-exchangecolumn chromatographies (e.g., cation exchange or anion exchangechromatographies).

In embodiments, the magnetic resin beads of the charge-based, fluidicpurification apparatus have a diameter of about 0.2 micron to about 200micron. The diameter of the magnetic resin beads may depend on thebiological product being purified and the flow rate of the process.Moreover, the magnetic resin beads may have a concentration ranging from0.01% to 25% by weight. For example, the concentration of the magneticresin beads may be about 1% by weight. In other examples, the charge orelectrostatic association capacity of the magnetic resin beads is afunction of the bead concentration, surface area-to-volume ratio,surface charge density, net charge, or any combination thereof. In yetother examples, the magnetic resin beads may be solid, porous,nanoporous, microporous, or any combination thereof.

The at least one hybrid fluidic device or chip of the charge-based,fluidic purification apparatus may refer to, for example, amicrofluidic, a mesofluidic, a millifluidic, a macrofluidic device orchip, or any combination thereof. In some examples, the fluidic deviceor chip is a hybrid microfluidic device or chip, for example, amicrofluidic device that combines the functionality of cross-flow fluiddynamics with magnetophoretic and dielectrophoretic capabilities,wherein the cross-flow fluid dynamics are governed by the microchanneldesign, the magnetophoresis is accomplished via an external magneticfield, and the dielectrophoresis is accomplished via a dielectrophoreticelectrode. In aspects, the combination of the dielectrophoreticelectrode and the external magnetic field is used to manipulate the flowpath of the magnetic resin beads in the cross-flow microchannel toenable efficient purification at high flow rates (e.g., greater than 0.5mL/min), a phenomenon not currently realized in the field ofmicrofluidics in which flow rates are traditionally limited to μL/hr orμL/min. In other examples, the hybrid fluidic is a microfluidic devicethat combines the functionality of cross-flow fluid dynamics withmagnetophoretic and acoustophoretic capabilities, wherein the cross-flowfluid dynamics are governed by the microchannel design, themagnetophoresis is accomplished via an external magnetic field, and theacoustophoresis is accomplished via a piezoelectric transducer orcrystal. In aspects, the combination of the piezoelectric transducer andthe external magnetic field is used to manipulate the flow path of themagnetic resin beads in the cross-flow microchannel to enable efficientpurification at high flow rates (e.g., greater than 0.5 mL/min), aphenomenon not currently realized in the field of microfluidics in whichflow rates are traditionally limited to μL/hr or μL/min.

In embodiments, the charge-based, fluidic purification module furtherincludes at least one tangential flow filtration system operated infed-batch or perfusion mode to concentrate and buffer exchange thefraction containing the biological product.

In embodiments described herein, the magnetic resin beads of one or bothof the first (affinity-based, fluidic purification) and/or second(charged-based, fluidic purification) modules are recycled and re-used.For example, the beads may be re-used at least 2, 3, 4, or more timesfor purifying a biological product.

Alternatively, the process described herein includes continuouslytransferring the fraction containing the biological product from the atleast one first outlet of the first module to a second module having atleast one inlet for receiving flow from the at least one first outlet ofthe first module, and the second module comprises a free-flowelectrophoresis apparatus. The free-flow electrophoresis apparatushaving a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and an aqueous ionic solution, may be used in lieu of or inaddition to the charge-based, magnetic purification module(s) to purifythe biological product (e.g., a monoclonal antibody).

In examples, the solution contacting surfaces of the two parallel platescomprise glass, ceramic, plastic, or any combination thereof. In someexamples, the aqueous ionic solution may give rise to a pH gradientacross the main separation channel. In other examples, the aqueous ionicsolution may confer constant pH across the main separation channel.

In embodiments, the free-flow electrophoresis apparatus has at least onefluidic device comprising a fluidic channel created between two parallelplates, an electric field or electric field gradient orthogonal to thefluid flow direction, and a pH gradient. In examples, the isoelectricpoint-based, fluidic purification module includes at least one firstfluidic device comprising a fluidic channel created between two parallelplates, an electric field or electric field gradient orthogonal to thefluid flow direction, and a coarse pH gradient across the mainseparation channel (in examples, a coarse pH gradient may be a pH rangefrom about 2 to about 10); and at least one second fluidic devicecomprising a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and a fine pH gradient across the main separation channel (inexamples, a fine pH gradient may be a pH range from about 5 to about 8).In examples, additional, subsequent fluidic devices or chips comprisinga fluidic channel created between two parallel plates and an electricfield or electric field gradient orthogonal to the fluid flow directionmay be used to enable further refining of the pH gradient across themain separation channel (e.g., a pH range from about 7.1 to about 7.6).

In other embodiments, the free-flow electrophoresis apparatus has atleast one fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and no pH gradient to operate ina zone electrophoresis or charge separating mode of operation. Inexamples, the isoelectric point-based, fluidic purification moduleincludes at least one first fluidic device comprising a fluidic channelcreated between two parallel plates, an electric field or electric fieldgradient orthogonal to the fluid flow direction, and constant basic pH(e.g., a pH of greater than 7); and at least one second fluidic devicecomprising a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and a constant acidic pH (e.g., a pH of less than 7).

In other embodiments, the free-flow electrophoresis apparatus has atleast one fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and both an acidic pH gradientand a basic pH gradient separated by a spacer solution (e.g. NaClsolution) to operate in an isotachophoresis mode of operation.

In other embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first free-flow electrophoresis apparatuscomprising a fluidic channel created between two parallel plates and anelectric field or electric field gradient orthogonal to the fluid flowdirection, and at least one second free-flow electrophoresis apparatuscomprising a fluidic channel created between two parallel plates and anelectric field or electric field gradient orthogonal to the fluid flowdirection, wherein each device connected in series and is capable ofoperating in an independent mode of operation to enable purification.For example, the at least one first free-flow electrophoresis apparatusmay operate in an isoelectric focusing mode and the at least one secondfree-flow electrophoresis apparatus may operate in an isotachophoresismode to increase separation resolution.

In other embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first fluidic device comprising fluidicchannel having at least one dielectrophoretic electrode capable ofinducing a defined, unidirectional force; at least one second free-flowelectrophoresis apparatus comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and a coarse pH gradient acrossthe main separation channel (e.g., a pH range from about 2 to about 10);and at least one third free-flow electrophoresis apparatus comprising afluidic channel created between two parallel plates, an electric fieldor electric field gradient orthogonal to the fluid flow direction, and afine pH gradient across the main separation channel (e.g., a pH rangefrom about 5 to about 8). In examples, additional, subsequent fluidicdevices or chips comprising a fluidic channel created between twoparallel plates and an electric field or electric field gradientorthogonal to the fluid flow direction may be used to enable furtherrefining of the pH gradient across the main separation channel (e.g., apH range from about 7.1 to about 7.6).

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least two electrodes (e.g. platinum wireelectrodes) to function as an anode or a cathode.

In embodiments, the backpressure within the isoelectric point-basedfluidic purification apparatus is dependent on the channel geometry anddimensions, the inlet and outlet opening and/or tubing diameters, andthe input flow rate. In examples, the backpressure ranges from about 0.5psi to about 10 psi. In some examples, the backpressure is controlledby, for example, without intent to be limiting, a needle valve.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least one de-bubbler system tocontinuously remove O₂ and H₂ gas bubbles that evolve in the electrodechannels under applied voltage. In some embodiments, removal ofelectrolysis bubbles is essential to enable continuous operation forsubstantially long periods of time. In examples, the de-bubbler systemutilizes a hydrophobic PTFE membrane to create a water-tight seal atopthe electrode channel that permits continuous removal of electrolysisbubbles at the point of generation by exposure to a vacuum system. Inexamples, the vacuum gauge pressure ranges from about −0.05 bar to about−0.4 bar.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises an active cooling system or heat sink (e.g.,a Peltier device, a thermal chuck with a circulating water/propyleneglycol jacket) to enable temperature control and Joule heat dissipation.For example, the active cooling system may control cooling and/or heatdissipation in the range from about 4° C. to about 50° C., preferablyfrom about 4° C. to about 37° C. Ideally, when isolating a biologicalproduct (e.g., a monoclonal antibody), the temperature is maintained atabout 10° C. to about 25° C. In examples, the active cooling systemcomprises an aluminum thermal chuck containing a chilled, circulatingwater/propylene glycol jacket.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one buffer or ampholyte system.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one electrode solution. In some embodiments, the atleast one electrode solution comprises an electrolyte solutionconfigured to contact and enable the appropriate function of an anode ora cathode, for example, phosphoric acid and sodium hydroxide,respectively. In other embodiments, the at least one electrode solutioncomprises at least one ampholyte solution configured to contact andenable the appropriate function of an anode or a cathode, for example,Tris buffered saline flowing through the main separation channel, theanode channel, and the cathode channel.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one sensor or detector. In examples, the at least onesensor or detector is positioned in-line. In some examples, the at leastone sensor or detector includes, but is not limited to, a flow sensor, atemperature sensor, a conductivity sensor, a pH sensor, a refractiveindex detector, a UV detector, or a backpressure sensor.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one liquid circuit breaker or disconnect downstream ofthe device and upstream of the at least one in-line sensor or detectorto ensure the ability to perform sensing or detection in a voltage-freesolution.

The presently claimed process provides for a number of advantages overcurrent downstream methods and processes for purifying a biologicalproduct, for example, a protein or fragment thereof (a polypeptide), anantibody or fragment thereof, a cytokine, a chemokine, an enzyme, or agrowth factor. For example, without intent to be limiting, the processdescribed herein provides a continuous bioprocess for purifying amonoclonal antibody that maintains throughput and yield, whilesignificantly decreasing the production facility footprint, the timerequired for facility buildout and validation, the costs associated withfacility buildout, and capital equipment expenditure, when compared tothe traditional approaches of batch, single-use, or semi-continuousmonoclonal antibody manufacturing. The continuous bioprocessing asdescribed herein affords smaller, more streamlined equipment (e.g.,smaller bioreactor volumes and downstream bioprocess equipment) becausethe ability to operate continuously eliminates the need for the largeprocess equipment required for the centrifugation, depth filtration, andcolumn chromatography steps of traditional downstream bioprocessing,whose size is dictated by large bioreactor volumes. Further, thesmaller, more streamlined equipment operating continuously affords theuse of significantly smaller bioreactor(s) that produce monoclonalantibodies at steady-state. The continuous bioprocess as describedherein may also significantly decrease operating expenditures, overallbioprocess line downtime, and biological product loss when compared totraditional monoclonal antibody manufacturing approaches. Finally, theprocess described herein for purifying a biological product is conductedin a system with a footprint that occupies significantly less squarefootage than current techniques, without sacrificing product throughputor yield on a kilograms/year basis.

Advantages of the process and methods described herein include theability to remove large impurities (e.g., cells, cell debris, andaggregates) without membrane fouling or occlusion. For example, it isknown in the art that clarification of cells, cell debris and aggregatesfrom cell culture media with traditional filtration or tangential flowfiltration systems typically leads to fouling or occlusion of the filtermembrane, thus rendering these methodologies unsuitable as a means tocontinuously remove large impurities from a heterogeneous mixturecontaining a biological product over long-term continuous processing. Incontrast, the dynamic filtration apparatus described herein enablescontinuous removal of large impurities from a heterogeneous mixturecontaining a biological product without membrane fouling, as the activetarget region of the filter membrane is constantly being refreshed.Additionally, because the entire process of producing and purifying thebiological product may be continuous and can maintain a flow rate thatranges from about 0.1 mL/minute to about 50 mL/minute across theentirety of the process, the process equipment and overall processfootprint is able to have a significantly smaller footprint than currentstandard processes, without sacrificing product throughput or yield on akilogram/year basis. For example, the process for producing andpurifying a monoclonal antibody as described herein is operated with afootprint that occupies up to about 30,000 square feet. In contrast,current monoclonal antibody production and downstream processes requireat least 200,000 square feet. In examples, the process of purifying thebiological product has a flow rate that ranges from about 1 mL/minute toabout 10 mL/minute. In some examples, the flow rate of the step ofcontinuously removing large impurities from the heterogeneous mixtureranges from about 0.1 mL/minute to about 50 mL/minute. In otherexamples, the flow rate of the step of continuously removing largeimpurities from the heterogeneous mixture is equivalent to the flow ratefrom the bioreactor bleed line. In other examples, the process providesthat the flow rate of the step of continuously transferring the filtrateto a first module ranges from about 0.1 mL/minute to about 50 mL/minute.In yet other examples, the process provides that the flow rate of thestep of continuously transferring the fraction containing the biologicalproduct from the first outlet to a second module ranges from about 0.1mL/minute to about 50 mL/minute. Further, the ability for the process ofpurifying the biological product at a flow rate that ranges from about0.1 mL/minute to about 50 mL/minute with a hybrid microfluidic device, aphenomenon not currently realized in the field of microfluidics in whichflow rates are traditionally limited to μL/hr or μL/min.

An important advantage of the process and methods utilizing magneticresin beads (e.g. magnetic agarose) described herein includes that thesesystems do not require traditional stationary phase or packed resincolumns (e.g., for standard chromatographies) to be sanitized, recycledand/or regenerated. For example, these systems provide for recyclingand/or regeneration of the magnetic resin beads to create a limitlesssurface area of the magnetic resin beads during operation, and in turnprovides a continuous and cost-effective method. Put in another way, themodules described herein do not have a fixed binding or associationcapacity. In specific examples, the magnetic resin beads used duringpurification of the biological product, as described herein, areconstantly being recycled and regenerated, and therefore able to acceptflow from the previous step, either a dynamic filtration module or apurification module, without interruption of the flow from thebioreactor bleed line. Put another way, the modules described in thepresent invention do not have to be left idle in order to be sanitized,regenerated and/or recycled after running, as they are continuouslyundergoing these steps. The method differs from current continuouschromatographic methods, in that current column chromatography methodshave defined column capacity limitations due to resin packingconstraints and thus require column switching of multiple packed columnsto accept continuous input flow and enable regeneration and/or recyclingof the columns that have reached full capacity. Another advantage of themethods described herein includes the that the magnetic resin beads arenot packed into a stationary phase, rather the magnetic resin beads havemobility. This increases the surface area of the magnetic resin beadsthat is available for binding or association, as substantially more ofthe magnetic resin bead surface is exposed and free to bind.Additionally, the resin beads in a packed column are exposed to a highpressure differential in order to generate flow through the column anddamage from which is one of the reasons for less than desired columnlifetime. The mobile resin beads in the presently described inventionare subjected to substantially lower pressures which is much gentler onthe fragile beads, resulting in longer lifetimes. Additionally, thismobility makes the magnetic resin beads more likely to be completedregenerated and returned to their initial condition. This further addsto the cost-effectiveness of the methods described herein, as the resinis utilized more efficiently.

An important advantage of the process and methods utilizing free-flowelectrophoresis described herein includes that this system represents a“no product loss” process, in that, there is no need for the product tointeract with a resin or other purifying moieties, as the separationoccurs in aqueous solution according to the physicochemical propertiesof the target biological product via interaction with an electric field.Another advantage is observed in the resolving power of this approach,as a theoretically higher purity product is achievable when compared totraditional ion-exchange chromatographies. Additionally, the separationbased on intrinsic physicochemical properties extends the utility ofthis approach for the purification a plethora of biological products,including, but not limited to, a protein or fragment thereof (apolypeptide), an antibody or fragment thereof, a cytokine, a chemokine,an enzyme, a growth factor, an oligonucleotide, a virus, an adenovirus,an adeno-associated virus (AAV), or a lentivirus.

Further, the modular approach affords flexibility in process design toaccommodate a diverse range of biological products.

Continuous Process for Purifying a Biological Product Using a DynamicFiltration Module, an Affinity-Based TFF Purification Module, and atLeast One of a Charge-Based TFF Purification Module or an IsoelectricPoint-Based, Fluidic Purification Module

A continuous process for purifying a biological product is described;the process including continuously receiving, via an input line, aheterogeneous mixture containing the biological product, wherein thebiological product includes, but is not limited to, a protein orfragment thereof (a polypeptide), an antibody or fragment thereof, acytokine, a chemokine, an enzyme, or a growth factor. When purifying,the biological product (e.g., a monoclonal antibody) is substantiallypure when it is at least 60%, 70%, 80%, 90%, 95%, or even 99%, byweight, free from impurities (e.g., cells, cellular debris, aggregates,host cell proteins, undesired proteins and peptides, undesiredantibodies, undesired nucleic acids and oligonucleotides, viruses,salts, buffer components, surfactants, sugars, metallic contaminants,leachables, media components, and/or naturally-occurring organicmolecules with which it is naturally associated).

The process includes continuously removing large impurities from theheterogeneous mixture by dynamic filtration. Said dynamic filtrationprocess includes at least one dynamic filtration module thatcontinuously feeds the biological product from at least one output headin fluid communication with the input line to the dynamic filtrationmodule under negative pressure, thereby producing a filtrate comprisingthe biological product. The dynamic filtration module may furtherinclude at least one additional input line to supply a wash buffer via acoaxial output head or a separate monoaxial output head.

In embodiments, the process described herein includes purifying abiological product that is continuously produced in a bioreactor (e.g.,a fed-batch bioreactor, a perfusion bioreactor, a chemostat bioreactor).For example, the bioreactor includes a bioreactor feed line and anoutput bleed line to enable steady-state cell culture growth conditions,and the output bleed line functions as the input line to permitcontinuous fluid flow from the bioreactor to the dynamic filtrationmodule.

As described herein, the process of continuously removing largeimpurities from the heterogeneous mixture does not includecentrifugation, disk-stack centrifugation, depth filtration, staticfiltration, tangential flow filtration, a hydrocyclone, or anycombination thereof. The term “static filtration” refers to a process inwhich the heterogeneous mixture being filtered remains static, meaningfor example that the filter membrane (or depth filter) has a definedcapacity, and the rate of filtration decreases as the membrane reachesits capacity (e.g., membrane pores become occluded). In a “static” (asopposed to “dynamic”) filtration, the filter membrane remains stationary(does not move), and the flow (e.g., of the heterogeneous mixture)passes through the stationary filter membrane. These static filtrationmethods are common in the art and are simple and well-understood.

Unlike the static filtration methods commonly used in the art, theprocess herein describes a dynamic filtration module, wherein componentsof the dynamic filtration module move in a coordinated fashion (e.g.,the membrane moves or advances in accordance with the flow rate of theentire process) to enable filtration to occur continuously across afresh, unused target region of filter membrane. This eliminates membranefouling or occlusion and permits control over the filter cake packingand thickness during operation.

The dynamic filtration module includes a filter membrane roll, amembrane support structure, at least one support rod or roller, a vacuumline, a vacuum system, and at least one vacuum collection vessel.

In embodiments, the filter membrane roll includes a filter roll, whereinthe filter membrane, without intent to be limiting, comprisespolyethersulfone (PES), hydrophilic polysulfone, cellulose ester,cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF,polycarbonate, nylon, polytetrafluoroethylene (PTFE), hydrophilic PTFE,or any combination thereof.

The pore size of the rolled filter membrane depends on the biologicalproduct being purified. In examples, the rolled filter membrane has apore size in the range from 0.1 μm to 1 μm. Alternatively, the pore sizeis in the range from about 0.2 μm to about 0.45 μm, or the pore size isless than about 0.45 μm. In other examples, when purifying an antibody,the pore size of the rolled filter membrane is in the range of 0.2 μm toabout 0.45μm.

The filter membrane roll has a width from about 10 mm to about 600 mm.The width of the filter membrane roll, for example, may depend on thesize of the dynamic filtration system or the membrane support structure.

In embodiments, the filter membrane roll further functions as a feedreel that communicates with a collection reel, meaning the filtermembrane originates from pre-fabricated roll and spans to an initiallyempty collecting roll, thus creating a reel-to-reel system. In aspects,the dynamic filtration module includes a rolled filter membraneextending between a feed reel and a collection reel, the filter membranehaving an active target region that is configured to receive theheterogeneous mixture. In examples, the feed reel motion is governed bya Servo motor coupled with a gear box to limit rotations per minute(RPM) by a ratio of 200:1 to enable low membrane transport velocitieswith high torque. The collection reel motion is governed by a Servomotor coupled with a gear box to limit RPM by a ratio of 200:1 to enablelow membrane transport velocities with high torque. Further, the feedreel motor and the collection reel motor are controlled by a closed-loopcontroller that operates a feedback mechanism to ensure consistentvelocity with the constantly changing diameters of the filter membraneroll on both the feed reel and the collection reel during operation. Inexamples, the feed reel and the collection reel operate in the samedirection with equivalent velocities.

In embodiments, the transport velocity of the filter membrane rangesfrom about 0.1 mm/sec to about 100 mm/sec, preferably from about 0.1mm/sec to about 10 mm/sec.

The membrane support structure of the dynamic filtration module includesa mechanically smooth contact surface derived from a material having alow static coefficient of friction (e.g. PTFE) and an opening that hascontinuity with the vacuum line. As used herein, the “membrane supportstructure” refers to a fabricated component that provides structuralsupport to the active region of the filter membrane, to preventdeformation, as it traverses an area of negative pressure, resultingfrom the opening having continuity with the vacuum line. Further, asused herein, “mechanically smooth contact surface” refers to a surfacehaving a low static coefficient of friction, thus creating a lowfrictional force opposing transport of the filter membrane, especiallywhen wetted. The mechanically smooth contact surface may influence theease at which the filter membrane moves in a dynamic fashion. Themechanically smooth contact surface may also be measured in surfaceroughness, where the lower the value the smoother the surface. Moreover,since rougher surfaces have more friction between them than smoothersurfaces, the mechanically smooth contact surface, as used herein,refers to a surface having lower friction (i.e., a low staticcoefficient of friction).

In embodiments, the membrane support structure of the dynamic filtrationmodule includes an opening. The opening for example, may include a mesh,at least one slot, at least one hole, a frit, a porous material, or anycombination thereof. For example, the opening may include a series ofregularly or irregularly spaced elements (e.g., a mesh, at least oneslot, at least one hole, or any combination thereof). Moreover, theopening may include regularly spaced elements, for example the openingmay include a series of equally spaced, parallel slots. Additionally,the opening can include one grate (e.g., a series of regularly orirregularly spaced elements as described above). In other examples, theopening can include more than one grate, with each grate perpendicular.The opening can be a collection of irregular or regular elements (e.g.,a series of parallel slots). The opening can also include a mesh, whichare of split-thickness or of full-thickness and may or may not be inparallel rows. The elements of the opening (e.g., a mesh, at least oneslot, at least one hole, a frit, a porous material, or any combinationthereof) may be of any desired thickness. For example, without intent tobe limiting, the opening may include a mesh with a thickness of about0.25 mm to about 5 mm.

The membrane support structure of the dynamic filtration module includesa temperature control mechanism. The temperature control mechanismmaintains a temperature from 4° C. to 37° C. in the presence ofevaporative cooling. For example, during purification of an antibody,the temperature control mechanism maintains a temperature from 15° C. to37° C. Exemplary temperature control mechanisms include, but are notlimited to, single loop controllers, multi-loop controllers, closed loopcontrollers, PID controllers, Peltier devices, resistive heatingelements, and/or thermal chucks with circulating water jackets.

In embodiments, the at least one support rod or roller of the dynamicfiltration module has a mechanically smooth contact surface derived froma material having a low static coefficient of friction (e.g. PTFE, PFA).In examples, the dynamic filtration module includes at least one supportrod or roller with a mechanically smooth contact surface to stabilizethe motion of the filter membrane across the membrane support structure.

In embodiments, the dynamic filtration module includes at least oneoutput head for modulating flow of the heterogeneous mixture anddispensing the heterogeneous mixture onto the active target region ofthe filter membrane. In examples, the at least one output head is a tubeor a slot die.

In some embodiments, the dynamic filtration module further includes atleast one additional input line to supply a wash buffer via a coaxialoutput head, a separate monoaxial output head, a separate slot dieoutput head, or a slot die output head with multiple openings.

In some embodiments, the dynamic filtration module includes elementsknown in the coating and converting industry, for example, withoutintent to be limiting, active or passive edge guides, tension control(e.g. a dancer), break and tension detectors, or any combinationthereof.

In embodiments, the dynamic filtration module includes a vacuum systemhaving continuity with the membrane support structure to apply negativepressure across the active target region of the filter membrane, wherethe negative pressure allows for active transport of the filter membraneacross the membrane support structure and enables collection of thefiltrate containing the biological product. In examples, the vacuumsystem of the dynamic filtration module maintains a gauge pressure ofabout −0.05 bar to about −0.98 bar for continuous filtration.

In embodiments, the dynamic filtration module further includes at leastone vacuum collection vessel configured to collect the filtrate, and atleast one sensor or detector. In aspects described herein, during thepurification by dynamic filtration, the filtrate comprising thebiological product is fed under negative pressure into a vacuumcollection vessel capable of collecting from about 50 mL to about 100 L.In examples, the vacuum collection vessel capable of collecting thefiltrate is from about 1 L to about 10 L. In other examples, the vacuumcollection vessel capable of collecting the filtrate is from about 1 Lto about 50 L.

In embodiments, the process of continuously removing large impurities(e.g., cells, cell debris, and aggregates) from the heterogeneousmixture by dynamic filtration comprises a multiple stage filtration withat least two discrete rolled filter membranes with different pore sizes.In examples, this multiple stage dynamic filtration process includes atleast one first dynamic filtration apparatus having a rolled filtermembrane with a large pore size (e.g., 0.45 μm) in fluid communicationwith at least one second dynamic filtration apparatus having a rolledfilter membrane with a small pore size (e.g., 0.2 μm), thereby producinga filtrate comprising the biological product.

The process described herein includes continuously transferring thefiltrate to a first module capable of separating the solution into twoor more fractions including at least one fraction containing thebiological product. For example, separating the solution into two ormore fractions, may include at least one fraction containing thebiological product, and the at least one other fraction containing smallimpurities. As described herein, the first module comprises anaffinity-based TFF purification apparatus. The “affinity-based TFFpurification apparatus” refers to a purification technique based onutilizing molecular, conformational binding interactions (e.g.,ligand-receptor interactions) in which selective surface-immobilizedligands recognize and bind to the biological product to be purified withat least one tangential flow filtration system. In examples, the firstmodule has at least one first inlet and at least one first outlet and isconfigured to permit continuous fluid flow between the first inlet andthe first outlet.

In embodiments, the affinity-based TFF purification apparatus furtherincludes a suspension of resin beads. The surface of the resin beads,for example, without intent to be limiting, is coupled with Protein A,Protein G, Protein L, an antigenic protein, a protein, a receptor, anantibody, or an aptamer. Continuous purification of biological products(e.g., a monoclonal antibody) with affinity resin beads can avoid thecumbersome processing steps of traditional affinity columnchromatography (e.g., Protein A affinity chromatography).

In embodiments, the resin beads of the affinity-based TFF purificationapparatus have a diameter of about 10 micron to about 200 micron. Thediameter of the resin beads may depend on the biological product beingpurified and the flow rate of the process. Moreover, the resin beads mayhave a concentration ranging from 0.01% to 25% by weight. For example,the concentration of the resin beads may be about 1% to about 20% byweight. In other examples, the binding capacity of the resin beads is afunction of the bead concentration, surface area-to-volume ratio,affinity ligand density, or any combination thereof. In yet otherexamples, the resin beads may be solid, porous, nanoporous, microporous,or any combination thereof.

The at least one tangential flow filtration system of the affinity-basedTFF purification apparatus may refer to, for example, a tangential flow,high performance tangential flow, or cross-flow filtration system havingflat plate or hollow fiber membrane filtration geometries. In someexamples, the tangential flow filtration system comprises a hollow fibermembrane filter. In aspects, the hollow fiber membrane material isselected from PES, modified PES (mPES), or mixed cellulose ester (MCE).In some aspects, the hollow fiber membrane may be charged (e.g.,positively or negatively) or uncharged. In other aspects, the pore sizeof the hollow fiber membrane is selected from the range of about 10 kDato about 1 μm. In yet other aspects, the inner diameter of the hollowfiber membrane is selected from the range of about 0.5 mm to about 5 mm.

The process as described herein also includes continuously transferringthe fraction containing the biological product from the at least onefirst outlet of the first module to a second module having at least oneinlet for receiving flow from the at least one first outlet of the firstmodule, and the second module comprises a charge-based TFF purificationapparatus. The charge-based TFF purification apparatus” as used hereinincludes, for example, purifying biological molecules based on theirsurface charge, ionic character, electrostatic interactions, orisoelectric point with at least one tangential flow filtration system.As described herein, the charge-based TFF purification comprises apositive charge-based TFF purification apparatus, negative charge-basedTFF purification apparatus, or combinations thereof. In examples, thesecond module has at least one second inlet and at least one secondoutlet and is configured to permit continuous fluid flow between thesecond inlet and the second outlet.

In embodiments, the charge-based TFF purification apparatus furtherincludes a suspension of resin beads. The surface of the resin beads,for example, may comprise cationic or anionic functionality configuredto selectively associate with said biological product at a specific pHand ionic strength to enable positive charge-based purification ornegative charge-based purification, respectively. Continuouspurification of biological products (e.g., a monoclonal antibody) withionic resin beads can avoid the cumbersome processing steps oftraditional ion-exchange column chromatographies (e.g., cation exchangeor anion exchange chromatographies).

In embodiments, the resin beads of the charge-based TFF purificationapparatus have a diameter of about 10 micron to about 200 micron. Thediameter of the resin beads may depend on the biological product beingpurified and the flow rate of the process. Moreover, the resin beads mayhave a concentration ranging from 0.01% to 25% by weight. For example,the concentration of the resin beads may be about 1% to about 20% byweight. In other examples, the charge or electrostatic associationcapacity of the resin beads is a function of the bead concentration,surface area-to-volume ratio, surface charge density, net charge, or anycombination thereof.

The at least one tangential flow filtration system of the charge-basedTFF purification apparatus may refer to, for example, a tangential flow,high performance tangential flow, or cross-flow filtration system havingflat plate or hollow fiber membrane filtration geometries. In someexamples, the tangential flow filtration system comprises a hollow fibermembrane filter. In aspects, the hollow fiber membrane material isselected from PES, modified PES (mPES), or mixed cellulose ester (MCE).In some aspects, the hollow fiber membrane may be charged (e.g.,positively or negatively) or uncharged. In other aspects, the pore sizeof the hollow fiber membrane is selected from the range of about 10 kDato about 1 μm. In yet other aspects, the inner diameter of the hollowfiber membrane is selected from the range of about 0.5 mm to about 5 mm.

In embodiments described herein, the resin beads of one or both of thefirst (affinity-based TFF purification) and/or second (charged-based TFFpurification) modules are recycled and re-used. For example, the beadsmay be re-used at least 2, 3, 4, or more times for purifying abiological product.

Alternatively, the process described herein includes continuouslytransferring the fraction containing the biological product from the atleast one first outlet of the first module to a second module having atleast one inlet for receiving flow from the at least one first outlet ofthe first module, and the second module comprises a free-flowelectrophoresis apparatus. The free-flow electrophoresis apparatushaving a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and an aqueous ionic solution, may be used in lieu of or inaddition to the charge-based, magnetic purification module(s) to purifythe biological product (e.g., a monoclonal antibody).

In examples, the solution contacting surfaces of the two parallel platescomprise glass, ceramic, plastic, or any combination thereof. In someexamples, the aqueous ionic solution may give rise to a pH gradient. Inother examples, the aqueous ionic solution may confer constant pH.

In embodiments, the free-flow electrophoresis apparatus has at least onefluidic device comprising a fluidic channel created between two parallelplates, an electric field or electric field gradient orthogonal to thefluid flow direction, and a pH gradient. In examples, the isoelectricpoint-based, fluidic purification module includes at least one firstfluidic device comprising a fluidic channel created between two parallelplates, an electric field or electric field gradient orthogonal to thefluid flow direction, and a coarse pH gradient across the mainseparation channel (in examples, a coarse pH gradient may be a pH rangefrom about 2 to about 10); and at least one second fluidic devicecomprising a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and a fine pH gradient across the main separation channel (inexamples, a fine pH gradient may be a pH range from about 5 to about 8).In examples, additional, subsequent fluidic devices or chips comprisinga fluidic channel created between two parallel plates and an electricfield or electric field gradient orthogonal to the fluid flow directionmay be used to enable further refining of the pH gradient across themain separation channel (e.g., a pH range from about 7.1 to about 7.6).

In other embodiments, the free-flow electrophoresis apparatus has atleast one fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and no pH gradient to operate ina zone electrophoresis or charge separating mode of operation. Inexamples, the isoelectric point-based, fluidic purification moduleincludes at least one first fluidic device comprising a fluidic channelcreated between two parallel plates, an electric field or electric fieldgradient orthogonal to the fluid flow direction, and constant basic pH(e.g., a pH of greater than 7); and at least one second fluidic devicecomprising a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and a constant acidic pH (e.g., a pH of less than 7).

In other embodiments, the free-flow electrophoresis apparatus has atleast one fluidic device comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and both an acidic pH gradientand a basic pH gradient separated by a spacer solution (e.g. NaClsolution) to operate in an isotachophoresis mode of operation.

In other embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first free-flow electrophoresis apparatuscomprising a fluidic channel created between two parallel plates and anelectric field or electric field gradient orthogonal to the fluid flowdirection, and at least one second free-flow electrophoresis apparatuscomprising a fluidic channel created between two parallel plates and anelectric field or electric field gradient orthogonal to the fluid flowdirection, wherein each device connected in series and is capable ofoperating in an independent mode of operation to enable purification.For example, the at least one first free-flow electrophoresis apparatusmay operate in an isoelectric focusing mode and the at least one secondfree-flow electrophoresis apparatus may operate in an isotachophoresismode to increase separation resolution.

In other embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first fluidic device comprising fluidicchannel having at least one dielectrophoretic electrode capable ofinducing a defined, unidirectional force; at least one second free-flowelectrophoresis apparatus comprising a fluidic channel created betweentwo parallel plates, an electric field or electric field gradientorthogonal to the fluid flow direction, and a coarse pH gradient acrossthe main separation channel (e.g., a pH range from about 2 to about 10);and at least one third free-flow electrophoresis apparatus comprising afluidic channel created between two parallel plates, an electric fieldor electric field gradient orthogonal to the fluid flow direction, and afine pH gradient across the main separation channel (e.g., a pH rangefrom about 5 to about 8). In examples, additional, subsequent fluidicdevices or chips comprising a fluidic channel created between twoparallel plates and an electric field or electric field gradientorthogonal to the fluid flow direction may be used to enable furtherrefining of the pH gradient across the main separation channel (e.g., apH range from about 7.1 to about 7.6).

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least two electrodes (e.g. platinum wireelectrodes) to function as an anode or a cathode.

In embodiments, the backpressure within the isoelectric point-basedfluidic purification apparatus is dependent on the channel geometry anddimensions, the inlet and outlet opening and/or tubing diameters, andthe input flow rate. In examples, the backpressure ranges from about 0.5psi to about 10 psi. In some examples, the backpressure is controlledby, for example, without intent to be limiting, a needle valve.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least one de-bubbler system tocontinuously remove O₂ and H₂ gas bubbles that evolve in the electrodechannels under applied voltage. In some embodiments, removal ofelectrolysis bubbles is essential to enable continuous operation forsubstantially long periods of time. In examples, the de-bubbler systemutilizes a hydrophobic PTFE membrane to create a water-tight seal atopthe electrode channel that permits continuous removal of electrolysisbubbles at the point of generation by exposure to a vacuum system. Inexamples, the vacuum gauge pressure ranges from about −0.05 bar to about−0.4 bar.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises an active cooling system or heat sink (e.g.,a Peltier device, a thermal chuck with a circulating water/propyleneglycol jacket) to enable temperature control and Joule heat dissipation.For example, the active cooling system may control cooling and/or heatdissipation in the range from about 4° C. to about 50° C., preferablyfrom 4° C. to about 37° C. Ideally, when isolating a biological product(e.g., a monoclonal antibody), the temperature is maintained at about10° C. to about 25° C. In examples, the active cooling system comprisesan aluminum thermal chuck containing a chilled, circulatingwater/propylene glycol jacket.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one buffer or ampholyte system.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one electrode solution. In some embodiments, the atleast one electrode solution comprises an electrolyte solutionconfigured to contact and enable the appropriate function of an anode ora cathode, for example, phosphoric acid and sodium hydroxide,respectively. In other embodiments, the at least one electrode solutioncomprises at least one ampholyte solution configured to contact andenable the appropriate function of an anode or a cathode, for example,Tris buffered saline flowing through the main separation channel, theanode channel, and the cathode channel.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one sensor or detector. In examples, the at least onesensor or detector is positioned in-line. In some examples, the at leastone sensor or detector includes, but is not limited to, a flow sensor, atemperature sensor, a conductivity sensor, a pH sensor, a refractiveindex detector, a UV detector, or a backpressure sensor.

In embodiments, the isoelectric point-based, fluidic purification moduleincludes at least one liquid circuit breaker or disconnect downstream ofthe device and upstream of the at least one in-line sensor or detectorto ensure the ability to perform sensing or detection in a voltage-freesolution.

The presently claimed process provides for a number of advantages overcurrent downstream methods and processes for purifying a biologicalproduct, for example, a protein or fragment thereof (a polypeptide), anantibody or fragment thereof, a cytokine, a chemokine, an enzyme, or agrowth factor. For example, without intent to be limiting, the processdescribed herein provides a continuous bioprocess for purifying amonoclonal antibody that maintains throughput and yield, whilesignificantly decreasing the production facility footprint, the timerequired for facility buildout and validation, the costs associated withfacility buildout, and capital equipment expenditure, when compared tothe traditional approaches of batch, single-use, or semi-continuousmonoclonal antibody manufacturing. The continuous bioprocessing asdescribed herein affords smaller, more streamlined equipment (e.g.,smaller bioreactor volumes and downstream bioprocess equipment) becausethe ability to operate continuously eliminates the need for the largeprocess equipment required for the centrifugation, depth filtration, andcolumn chromatography steps of traditional downstream bioprocessing,whose size is dictated by large bioreactor volumes. Further, thesmaller, more streamlined equipment operating continuously affords theuse of significantly smaller bioreactor(s) that produce monoclonalantibodies at steady-state. The continuous bioprocess as describedherein may also significantly decrease operating expenditures, overallbioprocess line downtime, and biological product loss when compared totraditional monoclonal antibody manufacturing approaches. Finally, theprocess described herein for purifying a biological product is conductedin a system with a footprint that occupies significantly less squarefootage than current techniques, without sacrificing product throughputor yield on a kilograms/year basis.

Advantages of the process and methods described herein include theability to remove large impurities (e.g., cells, cell debris, andaggregates) without membrane fouling or occlusion. For example, it isknown in the art that clarification of cells, cell debris and aggregatesfrom cell culture media with traditional filtration or tangential flowfiltration systems typically leads to fouling or occlusion of the filtermembrane, thus rendering these methodologies unsuitable as a means tocontinuously remove large impurities from a heterogeneous mixturecontaining a biological product over long-term continuous processing. Incontrast, the dynamic filtration apparatus described herein enablescontinuous removal of large impurities from a heterogeneous mixturecontaining a biological product without membrane fouling, as the activetarget region of the filter membrane is constantly being refreshed.Additionally, because the entire process of producing and purifying thebiological product may be continuous and can maintain a flow rate thatranges from about 0.1 mL/minute to about 50 mL/minute across theentirety of the process, the process equipment and overall processfootprint is able to have a significantly smaller footprint than currentstandard processes, without sacrificing product throughput or yield on akilogram/year basis. For example, the process for producing andpurifying a monoclonal antibody as described herein is operated with afootprint that occupies up to about 30,000 square feet. In contrast,current monoclonal antibody production and downstream processes requireat least 200,000 square feet. In examples, the process of purifying thebiological product has a flow rate that ranges from about 1 mL/minute toabout 10 mL/minute. In some examples, the flow rate of the step ofcontinuously removing large impurities from the heterogeneous mixtureranges from about 0.1 mL/minute to about 50 mL/minute. In otherexamples, the flow rate of the step of continuously removing largeimpurities from the heterogeneous mixture is equivalent to the flow ratefrom the bioreactor bleed line. In other examples, the process providesthat the flow rate of the step of continuously transferring the filtrateto a first module ranges from about 0.1 mL/minute to about 50 mL/minute.In yet other examples, the process provides that the flow rate of thestep of continuously transferring the fraction containing the biologicalproduct from the first outlet to a second module ranges from about 0.1mL/minute to about 50 mL/minute.

An important advantage of the process and methods utilizing resin beads(e.g. agarose) described herein includes that these systems do notrequire traditional stationary phase or packed resin columns (e.g., forstandard chromatographies) to be sanitized, recycled and/or regenerated.For example, these systems provide for recycling and/or regeneration ofthe resin beads to create a limitless surface area of the resin beadsduring operation, and in turn provides a continuous and cost-effectivemethod. Put in another way, the modules described herein do not have afixed binding or association capacity. In specific examples, the resinbeads used during purification of the biological product, as describedherein, are constantly being recycled and regenerated, and thereforeable to accept flow from the previous step, either a dynamic filtrationmodule or a purification module, without interruption of the flow fromthe bioreactor bleed line. Put another way, the modules described in thepresent invention do not have to be left idle in order to be sanitized,regenerated and/or recycled after running, as they are continuouslyundergoing these steps. The method differs from current continuouschromatographic methods, in that current column chromatography methodshave defined column capacity limitations due to resin packingconstraints and thus require column switching of multiple packed columnsto accept continuous input flow and enable regeneration and/or recyclingof the columns that have reached full capacity. Another advantage of themethods described herein includes the that the resin beads are notpacked into a stationary phase, rather the resin beads have mobility.This increases the surface area of the resin beads that is available forbinding or association, as substantially more of the resin bead surfaceis exposed and free to bind. Additionally, the resin beads in a packedcolumn are exposed to a high pressure differential in order to generateflow through the column and damage from which is one of the reasons forless than desired column lifetime. The mobile resin beads in thepresently described invention are subjected to substantially lowerpressures which is much gentler on the fragile beads, resulting inlonger lifetimes. Additionally, this mobility makes the resin beads morelikely to be completed regenerated and returned to their initialcondition. This further adds to the cost-effectiveness of the methodsdescribed herein, as the resin is utilized more efficiently.

An important advantage of the process and methods utilizing free-flowelectrophoresis described herein includes that this system represents a“no product loss” process, in that, there is no need for the product tointeract with a resin or other purifying moieties, as the separationoccurs in aqueous solution according to the physicochemical propertiesof the target biological product via interaction with an electric field.Another advantage is observed in the resolving power of this approach,as a theoretically higher purity product is achievable when compared totraditional ion-exchange chromatographies. Additionally, the separationbased on intrinsic physicochemical properties extends the utility ofthis approach for the purification a plethora of biological products,including, but not limited to, a protein or fragment thereof (apolypeptide), an antibody or fragment thereof, a cytokine, a chemokine,an enzyme, a growth factor, an oligonucleotide, a virus, an adenovirus,an adeno-associated virus (AAV), or a lentivirus.

Further, the modular approach affords flexibility in process design toaccommodate a diverse range of biological products.

Dynamic Filtration Module

Provided herein is a dynamic filtration module for continuously removinglarge impurities from a biological product in a heterogeneous mixture,for example, a filtrate containing a biological product is generated byremoving cell, cells debris, and aggregates from a heterogeneous mixturederived from a bioreactor operating at steady-state (FIGS. 6A-6B and7A-7B). Unlike the static filtration methods commonly used in the art,the components of the dynamic filtration module move in a coordinatedfashion (e.g., the membrane moves or advances in accordance with theflow rate of the entire process) to enable filtration to occurcontinuously across a fresh, unused target region of filter membrane.This eliminates membrane fouling or occlusion and permits control overthe filter cake packing and thickness during operation.

The dynamic filtration module includes a filter membrane roll, amembrane support structure, at least one support rod or roller, at leastone vacuum line, a vacuum system, and at least one vacuum collectionvessel. As shown in FIGS. 6A-6B and 7A-7B, the feed reel comprises afilter membrane that is disposed on a filter membrane roll, wherein thefilter membrane, supported by two mechanically smooth support rods,passes over the mechanically smooth membrane support structure, whichincludes an opening. As the heterogeneous mixture is delivered from theoutput head to the active target region of the filter membrane, thefilter membrane continues to move and advance the filter membrane towardthe collection reel, while a vacuum line, in continuity with the openingof the membrane support structure, maintains a negative pressure,allowing separation and removal of the cells, cell debris, andaggregates, thus creating a filtrate containing the biological product.

The dynamic filtration module enables removal of large impurities (e.g.,cells, cell debris, and aggregates), from the heterogeneous mixture toyield a filtrate containing the biological product and associated smallimpurities (e.g., host cell proteins, undesired proteins and peptides,undesired antibodies, undesired nucleic acids and oligonucleotides,viruses, undesired nucleic acids or oligonucleotides, salts, buffercomponents, surfactants, sugars, metallic contaminants, leachables,media components, and/or naturally-occurring organic molecules withwhich it is naturally associated) without centrifugation, depthfiltration, static filtration, or any combination thereof.

The dynamic filtration module described herein provides for a smallfootprint and requires appropriate materials selection for tubing,connectors, the membrane support structure, and the filter membrane(e.g., polymer type, pore size) to enable filtration with high yield,low protein binding, and minimal solution contact and residence times.

The dynamic filtration module includes a rolled filter membraneextending between a feed reel and a collection reel, wherein the filtermembrane has an active target region that is configured to receive theheterogeneous mixture. In examples, the filter membrane of the filtermembrane roll is made of a suitable material, including, but not limitedto, polyethersulfone (PES), hydrophilic polysulfone, cellulose ester,cellulose acetate, polyvinylidene fluoride (PVDF), hydrophilic PVDF,polycarbonate, nylon, polytetrafluoroethylene (PTFE), or hydrophilicPTFE.

In embodiments, the pore size of the rolled filter membrane depends onthe biological product being purified. In examples, the rolled filtermembrane has a pore size in the range from 0.1 μm to 1 μm.Alternatively, the pore size is in the range from about 0.2 μm to about0.45 μm, or the pore size is less than about 0.45 μm. In other examples,when purifying an antibody, the pore size of the rolled filter membraneis in the range of 0.2 μm to about 0.45 μm.

In embodiments, the filter membrane roll has a width from about 10 mm toabout 600 mm. The width of the filter membrane roll, for example, maydepend on factors such as the size of the dynamic filtration system andthe size of the membrane support structure.

In embodiments, the filter membrane roll further functions as a feedreel that communicates with a collection reel, meaning the filtermembrane originates from pre-fabricated roll and spans to an initiallyempty collecting roll, thus creating a reel-to-reel system. Inoperation, the heterogeneous mixture is continuously applied from theoutput head to a fresh, unused region of the filter membrane (alsoreferred to herein as the active target region) created by the transportof the filter membrane as it is moved at an appropriate rate from thefeed reel to the collection reel, thus collecting the used portions offilter membrane. In examples, the feed reel motion is governed by aServo motor coupled with a gear box to limit rotations per minute (RPM)by a ratio of 200:1 to enable low membrane transport velocities withhigh torque. The collection reel motion is governed by a Servo motorcoupled with a gear box to limit RPM by a ratio of 200:1 to enable lowmembrane transport velocities with high torque. Further, the feed reelmotor and the collection reel motor are controlled by a closed-loopcontroller that operates a feedback mechanism to ensure consistentvelocity with the constantly changing diameters of the filter membraneroll on both the feed reel and the collection reel during operation. Inexamples, the feed reel and the collection reel operate in the samedirection with equivalent velocities. Other methods of filter membranetransport from the feed reel to the collection reel can be contemplatedby those of skill in the art of the coating and converting industry.

In embodiments, the filter membrane transport velocity within thedynamic filtration module is selected to enable high flow rates (highthroughput), while maintaining high yield (recovery). In examples, thetransport velocity of the filter membrane ranges from about 0.1 mm/secto about 100 mm/sec, preferably from about 0.1 mm/sec to about 10mm/sec.

Additionally, the dynamic filtration module includes a membrane supportstructure (FIG. 8) to support the active target region of the filtermembrane as it experiences negative pressure. The membrane supportstructure is positioned between the feed reel and the collection reel,has a mechanically smooth contact surface derived from a material havinga low static coefficient of friction (e.g. PTFE), and has an openingthat has continuity with the vacuum line. For example, as used herein,“mechanically smooth contact surface” refers to a surface having a lowstatic coefficient of friction, thus creating a low frictional forceopposing transport of the filter membrane, especially when wetted. Themechanically smooth contact surface may influence the ease at which thefilter membrane moves in a dynamic fashion. The mechanically smoothcontact surface may also be measured in surface roughness, where thelower the value the smoother the surface. Moreover, since roughersurfaces have more friction between them than smoother surfaces, themechanically smooth contact surface, as used herein, refers to a surfacehaving lower friction (i.e., a low static coefficient of friction).

In embodiments, the membrane support structure of the dynamic filtrationmodule includes an opening. The opening for example, may include a mesh,at least one slot, at least one hole, a frit, a porous material, or anycombination thereof. For example, the opening may include a series ofregularly or irregularly spaced elements (e.g., a mesh, at least oneslot, at least one hole, or any combinations thereof). Moreover, theopening may include regularly spaced elements, for example the openingmay include a series of equally spaced, parallel slots. Additionally,the opening can include one grate (e.g., a series of regularly orirregularly spaced elements as described above). In other examples, theopening can include more than one grate, with each grate perpendicular.The opening can be a collection of irregular or regular elements (e.g.,a series of parallel slots). The opening can also include a mesh, whichare of split-thickness or of full-thickness and may or may not be inparallel rows. The elements of the opening (e.g., a mesh, at least oneslot, at least one hole, a frit, a porous material, or any combinationsthereof) may be of any desired thickness. For example, without intent tobe limiting, the opening may include a mesh with a thickness of about0.25 mm to about 5 mm.

Additionally, temperature control of the membrane support structure andits connection to the at least one vacuum collection vessel tocounteract evaporative cooling is also provided, which avoids clogging,fouling, solution freezing, changes in solution viscosity, anddenaturation (or precipitation) of the biological product (e.g., aprotein or fragment thereof (a polypeptide), an antibody or fragmentthereof, a cytokine, a chemokine, an enzyme, or a growth factor). Thetemperature control mechanism maintains a temperature from about 4° C.to about 37° C. For example, during purification of an antibody, thetemperature control mechanism maintains a temperature from about 15° C.to about 37° C. Exemplary temperature control mechanisms include, butare not limited to, single loop controllers, multi-loop controllers,closed loop controllers, PID controllers, Peltier devices, resistiveheating elements, and/or thermal chucks with circulating water jackets.

In embodiments, the at least one support rod or roller of the dynamicfiltration module has a mechanically smooth contact surface derived froma material having a low static coefficient of friction (e.g. PTFE, PFA).For example, as used herein, “mechanically smooth contact surface”refers to a surface having a low static coefficient of friction, thecreating a low frictional force opposing transport of the filtermembrane. The mechanically smooth contact surface may influence the easeat which the filter membrane moves in a dynamic fashion. Themechanically smooth contact surface may also be measured in surfaceroughness, where the lower the value the smoother the surface. Moreover,since rougher surfaces have more friction between them than smoothersurfaces, the mechanically smooth contact surface, as used herein,refers to a surface having lower friction (i.e., a low staticcoefficient of friction). Alternatively, the at least one support rodmay further include a bearing, for example, a sleeve bearing with amechanically smooth contact surface to reduce friction and tension onthe filter membrane. Additionally, the at least one support rod orroller having a mechanically smooth contact surface may rotate to reduceand tension on the filter membrane.

In embodiments, the dynamic filtration module includes at least oneoutput head for modulating flow of the heterogeneous mixture anddispensing the heterogeneous mixture onto the active target region ofthe filter membrane. In examples, the at least one output head is a tubeor a slot die.

Within the dynamic filtration module, the input flow rate matches thebleed rate of the bioreactor operating at steady-state, wherein saidbleed rate confers reasonably high throughput (e.g., consistent with orgreater than traditional biopharmaceutical manufacturing throughput on akilogram/year basis). In specific examples, multiple heads may be usedto manage flow rate, as well as xy rastering or rθ rastering heads, withor without motion along the z-axis.

The dynamic filtration module incorporates the negative pressure of avacuum system, which as described herein, the pressure value may beselected to enable efficient filtration, while maintaining desiredfilter membrane transport mobility to achieve high throughput and yield.In embodiments, the vacuum system of the dynamic filtration modulemaintains a gauge pressure of about −0.05 bar to about −0.98 bar forcontinuous filtration.

In some embodiments, a wash zone is provided in addition and subsequentto the feed zone (e.g., the bioreactor bleed solution input line andoutput head dispensing area or filter membrane active target region).The wash zone comprises a wash buffer that is supplied from anadditional input line via a coaxial output head, a separate monoaxialoutput head, a separate slot die output head, or a slot die output headwith multiple opening.

In some embodiments, the dynamic filtration module includes elementsknown in the coating and converting industry, for example, withoutintent to be limiting, active or passive edge guides, tension control(e.g. a dancer), break and tension detectors, or any combinationthereof.

In embodiments, the process of continuously removing large impurities(e.g., cells, cell debris, and aggregates) from the heterogeneousmixture by dynamic filtration comprises a multiple stage filtration withat least two discrete rolled filter membranes with different pore sizes.In examples, this multiple stage dynamic filtration process includes atleast one first dynamic filtration apparatus having a rolled filtermembrane with a large pore size (e.g., 0.45 μm) in fluid communicationwith at least one second dynamic filtration apparatus having a rolledfilter membrane with a small pore size (e.g., 0.2 μm), thereby producinga filtrate comprising the biological product.

As described herein, the dynamic filtration module provides for yieldsof biological product that are comparable or higher than standardpurification (centrifugation) processes on a kilogram/year basis. Thedynamic filtration module also allows for the ability to feed the nextstep from the at least one vacuum collection vessel that may be undernegative pressure or allowed to equilibrate to atmospheric pressure.Materials incorporated and selected for the dynamic filtration moduleinclude connectors, tubing, filter membrane, membrane support structure,vacuum collection vessel(s), all of which, alone or in combination,minimize friction and yield loss due to protein adsorption and are knownby those skilled in the art.

Unlike the static filtration methods commonly used in the art, thecomponents of the dynamic filtration module move in a coordinatedfashion (e.g., the membrane moves or advances in accordance with theflow rate of the heterogeneous mixture from the input line) to enablecontinuous, unimpeded and unfouled filtration.

Affinity-Based, Magnetic Purification Module Having a Loop ConveyorSystem

Provided herein is an affinity-based, magnetic purification module forseparating a heterogeneous mixture into two or more fractions, at leastone fraction containing a biological product. The module includes atleast one inlet and at least one outlet configured to permit continuousfluid flow between the at least one inlet and the at least one outletand wherein the flow rate may be, for example, consistent and constantduring steady-state operation. Moreover, the affinity-based, magneticpurification module includes a suspension of affinity magnetic resinbeads, wherein the magnetic resin bead surface, without intent to belimiting, is coupled to Protein A, Protein G, Protein L, an antigenicprotein, a protein, a receptor, an antibody, or an aptamer configured toselectively bind said biological product.

The affinity-based, magnetic purification module includes a loopconveyor system comprising at least two transport vessels charged withaffinity magnetic resin beads that are configured to continuouslyreceive a heterogeneous mixture containing a biological product andsubsequently transport the resulting heterogeneous mixture containing abiological product, affinity magnetic resin beads, a buffer, or anycombination thereof; at least one external magnetic field to attract,and thus separate, said magnetic resin beads from the heterogeneousmixture to enable washing; at least one external magnetic field toattract, and thus separate, said magnetic resin beads from theheterogeneous mixture to enable elution of said biological product; atleast one external magnetic field to enable recycling of said magneticresin beads; at least one binding/wash buffer system; at least one lowpH elution buffer system; at least one magnetic resin bead regenerationbuffer system; at least one aspirator system to remove waste solutionfrom the at least two transport vessels; at least one collection vessel;at least one sensor or detector; and at least one fluid handling pump.

The equipment design of the affinity-based, magnetic purification module(FIGS. 14-15) enables for automated and continuous, biological magneticpurification, as compared to a batch process or a semi-continuousprocess and provides for a small footprint.

The magnetic field strength and field effect are dependent on thetransport vessel wall thickness and material type, proximity to the wallof the transport vessel on a loop conveyor track, magnetic resin beadsize, concentration, saturation magnetization, and magneticsusceptibility, and solution viscosity. In embodiments, the magneticfield strength ranges from about 0.01 Tesla to about 1 Tesla (e.g., upto about 1 Tesla). In examples, the magnetic field is generated by apermanent magnet (e.g., a Neodymium magnet). The permanent magnet may bepositioned within 5 mm, or preferably within 1 mm of the vessel wall. Inother examples, the magnetic field is generated by an electromagnet. Inyet other examples, mixing of the magnetic resin beads may beaccomplished by placing the at least one transport vessel between twoseparate and opposing magnetic fields that toggle between states of onand off.

As described herein, the affinity-based, magnetic purification moduleincludes a suspension of magnetic resin beads, in which theconcentration depends on the desired binding capacity or the desiredsolution viscosity. Alternatively, the affinity-based, magneticpurification module magnetic resin bead size is micron to sub-micron andis dependent on the binding capacity needs, which is a function of thesurface area-to-volume ratio required to enable adequate fluid dynamicsfor equilibration and affinity interactions, for example, solutionviscosity dependency and surface ligand density dependency,respectively.

The transport vessel number and size depends on input flow rate,magnetic resin bead binding capacity, and binding equilibration time.The transport vessel material and wall thickness are dependent on thestrength and close proximity of the magnetic field.

The material selection for the magnetic resin beads of theaffinity-based, magnetic purification module is important for negligibleleachables and to provide for robust stability to enable recycling andreuse. The magnetic resin beads may be solid, porous, nanoporous,microporous, or any combination thereof.

The binding/wash buffer is dependent on the biological product (e.g.,monoclonal antibody) of interest and small impurities to be removed.Other considerations for the binding/wash buffer including pH, ionicstrength, use of surfactants, and use of organic and/or inorganic saltsare also contemplated in the binding/wash buffer. Moreover, additionaltransport vessels and replicate conveyor track positions may also berequired to effectively wash.

The elution buffer is dependent on the binding affinity (e.g., strengthof the non-covalent interactions) between the magnetic resin beadsurface ligands and the biological product (e.g., monoclonal antibody)of interest. For example, the elution buffer may vary pH, ionicstrength, use of surfactants, use of organic and/or inorganic salts, useof multiple elution buffer compositions (e.g., to increase yield, andwhich might require additional transport vessels and replicate conveyortrack positions). Moreover, additional transport vessels and replicateconveyor track positions may also be required to effectively elute.

The dwell time for each stage in conveyor track progression is dependenton flow rate, equilibration times, and throughput volume. Moreover,depending on buffer composition and pH, viral inactivation and removalduring the wash steps is also contemplated, and would thus eliminate theneed for a separate viral inactivation and removal process step, forexample, when purifying a monoclonal antibody.

The affinity-based, magnetic purification module may include in-linesampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, optical density measurementdevices, UV detectors, RI detectors) may be used to enable feedbackcontrol mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the affinity-based,magnetic purification module utilizes a mobile affinity resin capable ofin situ regeneration and recycling to enable more efficient use of theresin and to enable continuous processing in a small-footprint withoutconcerns of traditional column capacity limitations.

Affinity-Based, Magnetic Purification Module Having a Pick and PlaceRobotics System

Provided herein is an affinity-based, magnetic purification module forseparating a mixture into two or more fractions, at least one fractioncontaining a biological product. The module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation. Moreover, the affinity-based, magneticpurification module includes a suspension of affinity magnetic resinbeads, wherein the magnetic resin bead surface, without intent to belimiting, is coupled to Protein A, Protein G, Protein L, an antigenicprotein, a protein, a receptor, an antibody, or an aptamer configured toselectively bind said biological product.

The affinity-based, magnetic purification module includes a pick andplace robotics system comprising at least two transport vessels chargedwith affinity magnetic resin beads that are configured to continuouslyreceive a mixture containing a biological product and subsequentlytransport the resulting heterogeneous mixture containing a biologicalproduct, affinity magnetic resin beads, a buffer, or any combinationthereof; at least one external magnetic field to attract, and thusseparate, said magnetic resin beads from the heterogeneous mixture toenable washing; at least one external magnetic field to attract, andthus separate, said magnetic resin beads from the heterogeneous mixtureto enable elution of said biological product; at least one externalmagnetic field to enable recycling of said magnetic resin beads; atleast one binding/wash buffer system; at least one low pH elution buffersystem; at least one magnetic resin bead regeneration buffer system; atleast one aspirator system to remove waste solution from the at leasttwo transport vessels; at least one collection vessel; at least onesensor or detector; and at least one fluid handling pump.

The equipment design of the affinity-based, magnetic purification module(FIG. 18) enables for automated and continuous, biological magneticpurification, as compared to a batch process or a semi-continuousprocess and provides for a small footprint.

The magnetic field strength and field effect are dependent on thetransport vessel wall thickness and material type, proximity to the wallof the placed transport vessel, magnetic resin bead size, concentration,saturation magnetization, and magnetic susceptibility, and solutionviscosity. In embodiments, the magnetic field strength ranges from about0.01 Tesla to about 1 Tesla (e.g., up to about 1 Tesla). In examples,the magnetic field is generated by a permanent magnet (e.g., a Neodymiummagnet). The permanent magnet may be positioned within 5 mm, orpreferably within 1 mm of the vessel wall. In other examples, themagnetic field is generated by an electromagnet. In yet other examples,mixing of the magnetic resin beads may be accomplished by placing the atleast one transport vessel between two separate and opposing magneticfields that toggle between states of on and off.

As described herein, the affinity-based, magnetic purification moduleincludes a suspension of magnetic resin beads, in which theconcentration depends on the desired binding capacity or the desiredsolution viscosity. Alternatively, the affinity-based, magneticpurification module magnetic resin bead size is micron to sub-micron andis dependent on the binding capacity needs, which is a function of thesurface area-to-volume ratio required to enable adequate fluid dynamicsfor equilibration and affinity interactions, for example, solutionviscosity dependency and surface ligand density dependency,respectively.

The transport vessel number and size depends on input flow rate,magnetic resin bead binding capacity, and binding equilibration time.The transport vessel material and wall thickness are dependent on thestrength and close proximity of the magnetic field.

The material selection for the magnetic resin beads of theaffinity-based, magnetic purification module is important for negligibleleachables and to provide for robust stability to enable recycling andreuse. The magnetic resin beads may be solid, porous, nanoporous,microporous, or any combination thereof.

The binding/wash buffer is dependent on the biological product (e.g.,monoclonal antibody) of interest and small impurities to be removed.Other considerations for the binding/wash buffer including pH, ionicstrength, use of surfactants, and use of organic and/or inorganic saltsare also contemplated in the binding/wash buffer. Moreover, additionaltransport vessels and replicate pick and place positions may also berequired to effectively wash.

The elution buffer is dependent on the binding affinity (e.g., strengthof the non-covalent interactions) between the magnetic resin beadsurface ligands and the biological product (e.g., monoclonal antibody)of interest. For example, the elution buffer may vary pH, ionicstrength, use of surfactants, use of organic and/or inorganic salts, useof multiple elution buffer compositions (e.g., to increase yield, andwhich might require additional transport vessels and replicate conveyortrack positions). Moreover, additional transport vessels and replicatepick and place positions may also be required to effectively elute.

The dwell time for each stage in the progression of the transport vesselthrough the pick and place process is dependent on flow rate,equilibration times, and throughput volume. Moreover, depending onbuffer composition and pH, viral inactivation and removal during thewash steps is also contemplated, and would thus eliminate the need for aseparate viral inactivation and removal process step, for example, whenpurifying a monoclonal antibody.

The affinity-based, purification module may include in-line samplingports for in-process analytical testing and/or in-line analyticalmeasurement techniques. Further, the in-line analytical measurementtechniques (e.g., flow sensors, optical density measurement devices, UVdetectors, RI detectors) may be used to enable feedback controlmechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the affinity-based,magnetic purification module utilizes a mobile affinity resin capable ofin situ regeneration and recycling to enable more efficient use of theresin and to enable continuous processing in a small-footprint withoutconcerns of traditional column capacity limitations.

Positive Charge-Based, Magnetic Purification Module Having a LoopConveyor System

As described herein, a positive charge-based, magnetic purificationmodule for separating a mixture into two or more fractions, at least onefraction containing a biological product is included. The positivecharge-based, magnetic purification module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation. Moreover, the positive charge-based, magneticpurification module includes a suspension of cationic magnetic resinbeads, wherein the magnetic resin bead surface comprises cationicfunctionality configured to selectively associate with said biologicalproduct at a specific pH and ionic strength.

The positive charge-based, magnetic purification module includes a loopconveyor system comprising at least two transport vessels charged withcationic magnetic resin beads that are configured to continuouslyreceive a mixture containing a biological product and subsequentlytransport the resulting heterogeneous mixture containing a biologicalproduct, cationic magnetic resin beads, a buffer, or any combinationthereof; at least one external magnetic field to attract, and thusseparate, said magnetic resin beads from the heterogeneous mixture toenable washing; at least one external magnetic field to attract, andthus separate, said magnetic resin beads from the heterogeneous mixtureto enable dissociation of said biological product; at least one externalmagnetic field to enable recycling of said magnetic resin beads; atleast one association/wash buffer system, at least one dissociationbuffer system; at least one magnetic resin bead regeneration buffersystem; at least one aspirator system to remove waste solution from theat least two transport vessels; at least one collection vessel; at leastone sensor or detector; and at least one fluid handling pump.

The equipment design for the positive charge-based, magneticpurification module (FIGS. 16-17) enables for automated and continuous,biological magnetic purification as compared to a batch process or asemi-continuous process and provides for a small footprint.

The magnetic field strength and field effect are dependent on thetransport vessel wall thickness and material type, proximity to the wallof the transport vessel on a loop conveyor track, magnetic resin beadsize, concentration, saturation magnetization, and magneticsusceptibility, and solution viscosity. In embodiments, the magneticfield strength ranges from about 0.01 Tesla to about 1 Tesla (e.g., upto about 1 Tesla). In examples, the magnetic field is generated by apermanent magnet (e.g., a Neodymium magnet). The permanent magnet may bepositioned within 5 mm, or preferably within 1 mm of the vessel wall. Inother examples, the magnetic field is generated by an electromagnet. Inyet other examples, mixing of the magnetic resin beads may beaccomplished by placing the at least one transport vessel between twoseparate and opposing magnetic fields that toggle between states of onand off.

As described herein, the positive charge-based, magnetic purificationmodule includes a suspension of magnetic resin beads, in which theconcentration depends on the desired charge or electrostatic associationcapacity or the desired solution viscosity. Alternatively, the positivecharge-based, magnetic purification module magnetic resin bead size ismicron to sub-micron and is dependent on the charge or electrostaticassociation capacity needs, which is a function of the surfacearea-to-volume ratio required to enable adequate fluid dynamics forequilibration and charge or electrostatic interactions, for example,solution viscosity dependency and surface charge density dependency,respectively.

The transport vessel number and size depends on input flow rate,magnetic resin bead charge or electrostatic association capacity, andassociation equilibration time. The transport vessel material and wallthickness is dependent on the strength and close proximity of themagnetic field.

The material selection for the magnetic resin beads of the positivecharge-based, magnetic purification module is important for negligibleleachables and to provide for robust stability to enable recycling andreuse. The magnetic resin beads may be solid, porous, nanoporous,microporous, or any combination thereof.

Within the positive charge-based, magnetic purification module, thecationic surface selection is an important consideration and may includecationic polymers, net positively charged peptides or proteins, aminefunctionality. Further, the cationic surface selection is based onachieving appropriate electrostatic interactions and stability betweenthe positively charged bead surface and the biological product within adefined buffer (pH and ionic strength).

The association/wash buffer of the positive charge-based, magneticpurification module is dependent on the biological product of interest(e.g., a monoclonal antibody) and small impurities to be removed. ThepH, ionic strength, use of surfactants, and use of organic and/orinorganic salts is also contemplated in the association/wash buffer.Moreover, additional transport vessels and replicate conveyor trackpositions may also be required to effectively wash.

The dissociation buffer of the positive charge-based, magneticpurification module is dependent on the strength of the electrostaticinteractions between the cationic magnetic resin bead surfacefunctionality and the biological product (e.g., monoclonal antibody) ofinterest. For example, the dissociation buffer may vary pH, ionicstrength, use of surfactants, use of organic and/or inorganic salts, useof multiple dissociation buffer compositions (e.g., to increase yield,and which might require additional transport vessels and replicateconveyor track positions). In other examples, multiple dissociationbuffers varying pH, ionic strength, or any combination thereof, areutilized sequentially to create a gradient dissociation effect.Moreover, additional transport vessels and replicate conveyor trackpositions may also be required to effectively dissociate.

The dwell time for each stage in conveyor track progression of thepositive charge-based, magnetic purification module is dependent on flowrate, equilibration times, and throughput volume. Moreover, depending onbuffer composition and pH, viral inactivation and removal during thewash steps is also contemplated, and would thus eliminate the need for aseparate viral inactivation and removal process step, for example, whenpurifying a monoclonal antibody.

The positive charge-based, purification module may include in-linesampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, optical density measurementdevices, UV detectors, RI detectors) may be used to enable feedbackcontrol mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the positivecharge-based, magnetic purification module utilizes a mobile affinityresin capable of in situ regeneration and recycling to enable moreefficient use of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Positive Charge-Based, Magnetic Purification Module Having a Pick andPlace Robotics System

As described herein, a positive charge-based, magnetic purificationmodule for separating a mixture into two or more fractions, at least onefraction containing a biological product is included. The positivecharge-based, magnetic purification module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation. Moreover, the positive charge-based, magneticpurification module includes a suspension of cationic magnetic resinbeads, wherein the magnetic resin bead surface comprises cationicfunctionality configured to selectively associate with said biologicalproduct at a specific pH and ionic strength.

The positive charge-based, magnetic purification module includes a pickand place robotics system comprising at least two transport vesselscharged with cationic magnetic resin beads that are configured tocontinuously receive a mixture containing a biological product andsubsequently transport the resulting heterogeneous mixture containing abiological product, cationic magnetic resin beads, a buffer, or anycombination thereof; at least one external magnetic field to attract,and thus separate, said magnetic resin beads from the heterogeneousmixture to enable washing; at least one external magnetic field toattract, and thus separate, said magnetic resin beads from theheterogeneous mixture to enable dissociation of said biological product;at least one external magnetic field to enable recycling of saidmagnetic resin beads; at least one association/wash buffer system, atleast one dissociation buffer system; at least one magnetic resin beadregeneration buffer system; at least one aspirator system to removewaste solution from the at least two transport vessels; at least onecollection vessel; at least one sensor or detector; and at least onefluid handling pump.

The equipment design for the positive charge-based, magneticpurification module (FIG. 19) enables for automated and continuous,biological magnetic purification as compared to a batch process or asemi-continuous process and provides for a small footprint.

The magnetic field strength and field effect are dependent on thetransport vessel wall thickness and material type, proximity to the wallof the transport vessel on a loop conveyor track, magnetic resin beadsize, concentration, saturation magnetization, and magneticsusceptibility, and solution viscosity. In embodiments, the magneticfield strength ranges from about 0.01 Tesla to about 1 Tesla (e.g., upto about 1 Tesla). In examples, the magnetic field is generated by apermanent magnet (e.g., a Neodymium magnet). The permanent magnet may bepositioned within 5 mm, or preferably within 1 mm of the vessel wall. Inother examples, the magnetic field is generated by an electromagnet. Inyet other examples, mixing of the magnetic resin beads may beaccomplished by placing the at least one transport vessel between twoseparate and opposing magnetic fields that toggle between states of onand off.

As described herein, the positive charge-based, magnetic purificationmodule includes a suspension of magnetic resin beads, in which theconcentration depends on the desired charge or electrostatic associationcapacity or the desired solution viscosity. Alternatively, the positivecharge-based, magnetic purification module magnetic resin bead size ismicron to sub-micron and is dependent on the charge or electrostaticassociation capacity needs, which is a function of the surfacearea-to-volume ratio required to enable adequate fluid dynamics forequilibration and charge or electrostatic interactions, for example,solution viscosity dependency and surface charge density dependency,respectively.

The transport vessel number and size depends on input flow rate,magnetic resin bead charge or electrostatic association capacity, andassociation equilibration time. The transport vessel material and wallthickness is dependent on the strength and close proximity of themagnetic field.

The material selection for the magnetic resin beads of the positivecharge-based, magnetic purification module is important for negligibleleachables and to provide for robust stability to enable recycling andreuse. The magnetic resin beads may be solid, porous, nanoporous,microporous, or any combination thereof.

Within the positive charge-based, magnetic purification module, thecationic surface selection is an important consideration and may includecationic polymers, net positively charged peptides or proteins, aminefunctionality. Further, the cationic surface selection is based onachieving appropriate electrostatic interactions and stability betweenthe positively charged bead surface and the biological product within adefined buffer (pH and ionic strength).

The association/wash buffer of the positive charge-based, magneticpurification module is dependent on the biological product of interest(e.g., a monoclonal antibody) and small impurities to be removed. ThepH, ionic strength, use of surfactants, and use of organic and/orinorganic salts is also contemplated in the association/wash buffer.Moreover, additional transport vessels and replicate pick and placepositions may also be required to effectively wash.

The dissociation buffer of the positive charge-based, magneticpurification module is dependent on the strength of the electrostaticinteractions between the cationic magnetic resin bead surfacefunctionality and the biological product (e.g., monoclonal antibody) ofinterest. For example, the dissociation buffer may vary pH, ionicstrength, use of surfactants, use of organic and/or inorganic salts, useof multiple dissociation buffer compositions (e.g., to increase yield,and which might require additional transport vessels and replicateconveyor track positions). In other examples, multiple dissociationbuffers varying pH, ionic strength, or any combination thereof, areutilized sequentially to create a gradient dissociation effect.Moreover, additional transport vessels and replicate pick and placepositions may also be required to effectively dissociate.

The dwell time for each stage in the progression of the transport vesselthrough the pick and place process of the positive charge-based,magnetic purification module is dependent on flow rate, equilibrationtimes, and throughput volume. Moreover, depending on buffer compositionand pH, viral inactivation and removal during the wash steps is alsocontemplated, and would thus eliminate the need for a separate viralinactivation and removal process step, for example, when purifying amonoclonal antibody.

The positive charge-based, magnetic purification module may includein-line sampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, optical density measurementdevices, UV detectors, RI detectors) may be used to enable feedbackcontrol mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the positivecharge-based, magnetic purification module utilizes a mobile affinityresin capable of in situ regeneration and recycling to enable moreefficient use of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Negative Charge-Based, Magnetic Purification Module Having a LoopConveyor System

As described herein, a negative charge-based, magnetic purificationmodule for separating a mixture into two or more fractions, at least onefraction containing a biological product is included. The negativecharge-based, magnetic purification module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation. Moreover, the negative charge-based, magneticpurification module includes a suspension of anionic magnetic resinbeads, wherein the magnetic resin bead surface comprises anionicfunctionality configured to selectively associate with said biologicalproduct at a specific pH and ionic strength.

The negative charge-based, magnetic purification module includes a loopconveyor system comprising at least two transport vessels charged withanionic magnetic resin beads that are configured to continuously receivea mixture containing a biological product and subsequently transport theresulting heterogeneous mixture containing a biological product, anionicmagnetic resin beads, a buffer, or any combination thereof; at least oneexternal magnetic field to attract, and thus separate, said magneticresin beads from the heterogeneous mixture to enable washing; at leastone external magnetic field to attract, and thus separate, said magneticresin beads from the heterogeneous mixture to enable dissociation ofsaid biological product; at least one external magnetic field to enablerecycling of said magnetic resin beads; at least one association/washbuffer system; at least one dissociation buffer system; at least onemagnetic resin bead regeneration buffer system; at least one aspiratorsystem to remove waste solution from the at least two transport vessels;at least one collection vessel; at least one sensor or detector; and atleast one fluid handling pump.

The equipment design for the negative charge-based, magneticpurification module (FIGS. 16-17) enables for automated and continuous,biological magnetic purification as compared to a batch process or asemi-continuous process and provides for a small footprint.

The magnetic field strength and field effect are dependent on thetransport vessel wall thickness and material type, proximity to the wallof the transport vessel on a loop conveyor track, magnetic resin beadsize, concentration, saturation magnetization, and magneticsusceptibility, and solution viscosity. In embodiments, the magneticfield strength ranges from about 0.01 Tesla to about 1 Tesla (e.g., upto about 1 Tesla). In examples, the magnetic field is generated by apermanent magnet (e.g., a Neodymium magnet). The permanent magnet may bepositioned within 5 mm, or preferably within 1 mm of the vessel wall. Inother examples, the magnetic field is generated by an electromagnet. Inyet other examples, mixing of the magnetic resin beads may beaccomplished by placing the at least one transport vessel between twoseparate and opposing magnetic fields that toggle between states of onand off.

As described herein, the negative charge-based, magnetic purificationmodule includes a suspension of magnetic resin beads, in which theconcentration depends on the desired charge or electrostatic associationcapacity or the desired solution viscosity. Alternatively, the negativecharge-based, magnetic purification module magnetic resin bead size ismicron to sub-micron and is dependent on the charge or electrostaticassociation capacity needs, which is a function of the surfacearea-to-volume ratio required to enable adequate fluid dynamics forequilibration and charge or electrostatic interactions, for example,solution viscosity dependency and surface charge density dependency,respectively.

The transport vessel number and size depends on input flow rate,magnetic resin bead charge or electrostatic association capacity, andassociation equilibration time. The transport vessel material and wallthickness is dependent on the strength and close proximity of themagnetic field.

The material selection for the magnetic resin beads of the negativecharge-based, magnetic purification module is important for negligibleleachables and to provide for robust stability to enable recycling andreuse. The magnetic resin beads may be solid, porous, nanoporous,microporous, or any combination thereof.

Within the negative charge-based, magnetic purification module, theanionic surface selection is an important consideration and may includeanionic polymers, net negatively charged peptides or proteins,oligonucleotides, carboxyl functionality. Further, the selection isbased on achieving appropriate electrostatic interactions and stabilitybetween the negatively charged bead surface and the biological productwithin a defined buffer (pH and ionic strength).

The association/wash buffer of the negative charge-based, magneticpurification module is dependent on the biological product of interest(e.g., a monoclonal antibody) and small impurities to be removed. ThepH, ionic strength, use of surfactants, and use of organic and/orinorganic salts is also contemplated in the association/wash buffer.Moreover, additional transport vessels and replicate conveyor trackpositions may also be required to effectively wash.

The dissociation buffer of the negative charge-based, magneticpurification module is dependent on the strength of the electrostaticinteractions between the anionic magnetic resin bead surfacefunctionality and the biological product (e.g., monoclonal antibody) ofinterest. For example, the dissociation buffer may vary pH, ionicstrength, use of surfactants, use of organic and/or inorganic salts, useof multiple dissociation buffer compositions (e.g., to increase yield,and which might require additional transport vessels and replicateconveyor track positions). In other examples, multiple dissociationbuffers varying pH, ionic strength, or any combination thereof, areutilized sequentially to create a gradient dissociation effect.Moreover, additional transport vessels and replicate conveyor trackpositions may also be required to effectively dissociate.

The dwell time for each stage in conveyor track progression of thenegative charge-based, magnetic purification module is dependent on flowrate, equilibration times, and throughput volume. Moreover, depending onbuffer composition and pH, viral inactivation and removal during thewash steps is also contemplated, and would thus eliminate the need for aseparate viral inactivation and removal process step, for example, whenpurifying a monoclonal antibody.

The negative charge-based, magnetic purification module may includein-line sampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, optical density measurementdevices, UV detectors, RI detectors) may be used to enable feedbackcontrol mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the negativecharge-based, magnetic purification module utilizes a mobile affinityresin capable of in situ regeneration and recycling to enable moreefficient use of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Negative Charge-Based, Magnetic Purification Module Having a Pick andPlace Robotics System

As described herein, a negative charge-based, magnetic purificationmodule for separating a mixture into two or more fractions, at least onefraction containing a biological product is included. The negativecharge-based, magnetic purification module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation. Moreover, the negative charge-based, magneticpurification module includes a suspension of anionic magnetic resinbeads, wherein the magnetic resin bead surface comprises anionicfunctionality configured to selectively associate with said biologicalproduct at a specific pH and ionic strength.

The negative charge-based, magnetic purification module includes a pickand place robotics system comprising at least two transport vesselscharged with anionic magnetic resin beads that are configured tocontinuously receive a mixture containing a biological product andsubsequently transport the resulting heterogeneous mixture containing abiological product, anionic magnetic resin beads, a buffer, or anycombination thereof; at least one external magnetic field to attract,and thus separate, said magnetic resin beads from the heterogeneousmixture to enable washing; at least one external magnetic field toattract, and thus separate, said magnetic resin beads from theheterogeneous mixture to enable dissociation of said biological product;at least one external magnetic field to enable recycling of saidmagnetic resin beads; at least one association/wash buffer system; atleast one dissociation buffer system; at least one magnetic resin beadregeneration buffer system; at least one aspirator system to removewaste solution from the at least two transport vessels; at least onecollection vessel; at least one sensor or detector; and at least onefluid handling pump.

The equipment design for the negative charge-based, magneticpurification module (FIG. 19) enables for automated and continuous,biological magnetic purification as compared to a batch process or asemi-continuous process and provides for a small footprint.

The magnetic field strength and field effect are dependent on thetransport vessel wall thickness and material type, proximity to the wallof the transport vessel on a loop conveyor track, magnetic resin beadsize, concentration, saturation magnetization, and magneticsusceptibility, and solution viscosity. In embodiments, the magneticfield strength ranges from about 0.01 Tesla to about 1 Tesla (e.g., upto about 1 Tesla). In examples, the magnetic field is generated by apermanent magnet (e.g., a Neodymium magnet). The permanent magnet may bepositioned within 5 mm, or preferably within 1 mm of the vessel wall. Inother examples, the magnetic field is generated by an electromagnet. Inyet other examples, mixing of the magnetic resin beads may beaccomplished by placing the at least one transport vessel between twoseparate and opposing magnetic fields that toggle between states of onand off.

As described herein, the negative charge-based, magnetic purificationmodule includes a suspension of magnetic resin beads, in which theconcentration depends on the desired charge or electrostatic associationcapacity or the desired solution viscosity. Alternatively, the negativecharge-based, magnetic purification module magnetic resin bead size ismicron to sub-micron and is dependent on the charge or electrostaticassociation capacity needs, which is a function of the surfacearea-to-volume ratio required to enable adequate fluid dynamics forequilibration and charge or electrostatic interactions, for example,solution viscosity dependency and surface charge density dependency,respectively.

The transport vessel number and size depends on input flow rate,magnetic resin bead charge or electrostatic association capacity, andassociation equilibration time. The transport vessel material and wallthickness is dependent on the strength and close proximity of themagnetic field.

The material selection for the magnetic resin beads of the negativecharge-based, magnetic purification module is important for negligibleleachables and to provide for robust stability to enable recycling andreuse. The magnetic resin beads may be solid, porous, nanoporous,microporous, or any combination thereof.

Within the negative charge-based, magnetic purification module, theanionic surface selection is an important consideration and may includeanionic polymers, net negatively charged peptides or proteins,oligonucleotides, carboxyl functionality. Further, the selection isbased on achieving appropriate electrostatic interactions and stabilitybetween the negatively charged bead surface and the biological productwithin a defined buffer (pH and ionic strength).

The association/wash buffer of the negative charge-based, magneticpurification module is dependent on the biological product of interest(e.g., a monoclonal antibody) and small impurities to be removed. ThepH, ionic strength, use of surfactants, and use of organic and/orinorganic salts is also contemplated in the association/wash buffer.Moreover, additional transport vessels and replicate pick and placepositions may also be required to effectively wash.

The dissociation buffer of the negative charge-based, magneticpurification module is dependent on the strength of the electrostaticinteractions between the anionic magnetic resin bead surfacefunctionality and the biological product (e.g., monoclonal antibody) ofinterest. For example, the dissociation buffer may vary pH, ionicstrength, use of surfactants, use of organic and/or inorganic salts, useof multiple dissociation buffer compositions (e.g., to increase yield,and which might require additional transport vessels and replicateconveyor track positions). In other examples, multiple dissociationbuffers varying pH, ionic strength, or any combination thereof, areutilized sequentially to create a gradient dissociation effect.Moreover, additional transport vessels and replicate pick and placepositions may also be required to effectively dissociate.

The dwell time for each stage in the progression of the transport vesselthrough the pick and place process of the negative charge-based,magnetic purification module is dependent on flow rate, equilibrationtimes, and throughput volume. Moreover, depending on buffer compositionand pH, viral inactivation and removal during the wash steps is alsocontemplated, and would thus eliminate the need for a separate viralinactivation and removal process step, for example, when purifying amonoclonal antibody.

The negative charge-based, purification module may include in-linesampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, optical density measurementdevices, UV detectors, RI detectors) may be used to enable feedbackcontrol mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the negativecharge-based, magnetic purification module utilizes a mobile affinityresin capable of in situ regeneration and recycling to enable moreefficient use of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Affinity-Based Purification Module Having a Mechanical Rotary System

Provided herein is an affinity-based purification module for separatinga mixture into two or more fractions, at least one fraction containing abiological product. The module includes at least one inlet and at leastone outlet configured to permit continuous fluid flow between the atleast one inlet and the at least one outlet and wherein the flow ratemay be, for example, consistent and constant during steady-stateoperation. Moreover, the affinity-based purification module includes asuspension of affinity resin beads, wherein the resin bead surface,without intent to be limiting, is coupled to Protein A, Protein G,Protein L, an antigenic protein, a protein, a receptor, an antibody, oran aptamer configured to selectively bind said biological product.

The affinity-based purification module includes a lid system capable ofmotion along the z-axis having at least one gasketed lid, the at leastone gasketed lid comprising at least one inlet to introduce a gas toenable control of positive head pressure, at least one vent port toenable equilibration to atmospheric pressure, at least one inlet tointroduce a suspension of resin beads, at least one inlet to receive thefiltrate containing a biological product to enable binding, at least twoinlets to introduce a buffer system to disperse the resin beads toenable washing of, elution from, or regeneration of said resin beads; amechanical rotary system capable of motion in the xy-plane, for example,a carousel comprising at least two vessels charged with resin beads thatare configured to continuously receive a mixture containing a biologicalproduct and subsequently transport the resulting heterogeneous mixturecontaining a biological product, resin beads, a buffer, or anycombination thereof; a collection system capable of motion along thez-axis that interfaces with at least one of the at least two vessels ofthe mechanical rotary system to enable collection of waste, the fractioncontaining the biological product, or any combination thereof; at leastone gas; at least one binding/wash buffer system; at least one elutionbuffer system; at least one resin bead regeneration buffer system; atleast one collection vessel; at least one sensor or detector; and, atleast one fluid handling pump.

The equipment design of the affinity-based purification module (FIGS. 21and 23) enables for automated and continuous, biological purification,as compared to a batch process or a semi-continuous process and providesfor a small footprint.

As described herein, the affinity-based purification module includes asuspension of resin beads, in which the concentration depends on thedesired binding capacity or the desired solution viscosity.Alternatively, the affinity-based purification module resin bead size ismicron to sub-micron and is dependent on the binding capacity needs,which is a function of the surface area-to-volume ratio required toenable adequate fluid dynamics for equilibration and affinityinteractions, for example, solution viscosity dependency and surfaceligand density dependency, respectively.

The vessel number and size depends on input flow rate, resin beadbinding capacity, and binding equilibration time. The vessel material isselected to limit protein binding.

The filter or filter membrane material and pore size of the vesseldepends on the resin bead diameter and the biological product ofinterest. The filter or filter membrane materials is selected to limitprotein binding.

The material selection for the resin beads of the affinity-basedpurification module is important for negligible leachables and toprovide for robust stability to enable recycling and reuse. The resinbeads may be solid, porous, nanoporous, microporous, or any combinationthereof.

The binding/wash buffer is dependent on the biological product (e.g.,monoclonal antibody) of interest and small impurities to be removed.Other considerations for the binding/wash buffer including pH, ionicstrength, use of surfactants, and use of organic and/or inorganic saltsare also contemplated in the binding/wash buffer. Moreover, additionalvessels and replicate carousel positions may also be required toeffectively wash.

The elution buffer is dependent on the binding affinity (e.g., strengthof the non-covalent interactions) between the resin bead surface ligandsand the biological product (e.g., monoclonal antibody) of interest. Forexample, the elution buffer may vary pH, ionic strength, use ofsurfactants, use of organic and/or inorganic salts, use of multipleelution buffer compositions (e.g., to increase yield, and which mightrequire additional vessels and replicate carousel positions). Moreover,additional vessels and replicate carousel positions or additionalelutions may also be required to effectively elute.

The dwell time for each stage in the progression of the vessel throughthe rotary process is dependent on flow rate, equilibration times, andthroughput volume. Moreover, depending on buffer composition and pH,viral inactivation and removal during the wash steps is alsocontemplated, and would thus eliminate the need for a separate viralinactivation and removal process step, for example, when purifying amonoclonal antibody.

The affinity-based, purification module may include in-line samplingports for in-process analytical testing and/or in-line analyticalmeasurement techniques. Further, the in-line analytical measurementtechniques (e.g., flow sensors, optical density measurement devices, UVdetectors, RI detectors) may be used to enable feedback controlmechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the affinity-basedpurification module utilizes a mobile affinity resin capable of in situregeneration and recycling to enable more efficient use of the resin andto enable continuous processing in a small-footprint without concerns oftraditional column capacity limitations.

Positive Charge-Based Purification Module Having a Mechanical RotarySystem

As described herein, a positive charge-based purification module forseparating a mixture into two or more fractions, at least one fractioncontaining a biological product is included. The positive charge-basedpurification module includes at least one inlet and at least one outletconfigured to permit continuous fluid flow between the at least oneinlet and the at least one outlet and wherein the flow rate may be, forexample, consistent and constant during steady-state operation.Moreover, the positive charge-based purification module includes asuspension of cationic resin beads, wherein the resin bead surfacecomprises cationic functionality configured to selectively associatewith said biological product at a specific pH and ionic strength.

The positive charge-based purification module includes a lid systemcapable of motion along the z-axis having at least one gasketed lid, theat least one gasketed lid comprising at least one inlet to introduce agas to enable control of positive head pressure, at least one vent portto enable equilibration to atmospheric pressure, at least one inlet tointroduce a suspension of resin beads, at least one inlet to receive thefiltrate containing a biological product to enable association, at leasttwo inlets to introduce a buffer system to disperse the resin beads toenable washing of, dissociation from, or regeneration of said resinbeads; a mechanical rotary system capable of motion in the xy-plane, forexample, a carousel comprising at least two vessels charged with resinbeads that are configured to continuously receive a mixture containing abiological product and subsequently transport the resultingheterogeneous mixture containing a biological product, resin beads, abuffer, or any combination thereof; a collection system capable ofmotion along the z-axis that interfaces with at least one of the atleast two vessels of the mechanical rotary system to enable collectionof waste, the fraction containing the biological product, or anycombination thereof; at least one gas; at least one association/washbuffer system; at least one dissociation buffer system; at least oneresin bead regeneration buffer system; at least one collection vessel;at least one sensor or detector; and, at least one fluid handling pump.

The equipment design for the positive charge-based purification module(FIGS. 22-23) enables for automated and continuous, biologicalpurification as compared to a batch process or a semi-continuous processand provides for a small footprint.

As described herein, the positive charge-based purification moduleincludes cationic resin beads, in which the concentration depends on thedesired charge or electrostatic association capacity or the desiredsolution viscosity. Alternatively, the positive charge-basedpurification module resin bead size is micron to sub-micron and isdependent on the charge or electrostatic association capacity needs,which is a function of the surface area-to-volume ratio required toenable adequate fluid dynamics for equilibration and charge orelectrostatic interactions, for example, solution viscosity dependencyand surface charge density dependency, respectively.

The vessel number and size depends on input flow rate, resin bead chargeor electrostatic association capacity, and association equilibrationtime. The vessel material is selected to limit protein binding.

The filter or filter membrane material and pore size of the vesseldepends on the resin bead diameter and the biological product ofinterest. The filter or filter membrane materials is selected to limitprotein binding.

The material selection for the resin beads of the positive charge-basedpurification module is important for negligible leachables and toprovide for robust stability to enable recycling and reuse. The resinbeads may be solid, porous, nanoporous, microporous, or any combinationthereof.

Within the positive charge-based purification module, the cationicsurface selection is an important consideration and may include cationicpolymers, net positively charged peptides or proteins, aminefunctionality. Further, the selection is based on achieving appropriateelectrostatic interactions and stability between the positively chargedbead surface and the biological product within a defined buffer (pH andionic strength).

The association/wash buffer of the positive charge-based purificationmodule is dependent on the biological product of interest (e.g., amonoclonal antibody) and small impurities to be removed. The pH, ionicstrength, use of surfactants, and use of organic and/or inorganic saltsis also contemplated in the association/wash buffer. Moreover,additional vessels and replicate carousel positions may also be requiredto effectively wash.

The dissociation buffer of the positive charge-based purification moduleis dependent on the strength of the electrostatic interactions betweenthe anionic resin bead surface functionality and the biological product(e.g., monoclonal antibody) of interest. For example, the dissociationbuffer may vary pH, ionic strength, use of surfactants, use of organicand/or inorganic salts, use of multiple dissociation buffer compositions(e.g., to increase yield, and which might require additional vessels andreplicate carousel positions). In other examples, multiple dissociationbuffers varying pH, ionic strength, or any combination thereof, areutilized sequentially to create a gradient dissociation effect.Moreover, additional vessels and replicate carousel positions oradditional dissociations may also be required to effectively dissociate.

The dwell time for each stage in the progression of the vessel throughthe rotary process is dependent on flow rate, equilibration times, andthroughput volume. Moreover, depending on buffer composition and pH,viral inactivation and removal during the wash steps is alsocontemplated, and would thus eliminate the need for a separate viralinactivation and removal process step, for example, when purifying amonoclonal antibody.

The positive charge-based purification module may include in-linesampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, optical density measurementdevices, UV detectors, RI detectors) may be used to enable feedbackcontrol mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the positivecharge-based purification module utilizes a mobile affinity resincapable of in situ regeneration and recycling to enable more efficientuse of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Negative Charge-Based Purification Module Having a Mechanical RotarySystem

As described herein, a negative charge-based purification module forseparating a mixture into two or more fractions, at least one fractioncontaining a biological product is included. The negative charge-basedpurification module includes at least one inlet and at least one outletconfigured to permit continuous fluid flow between the at least oneinlet and the at least one outlet and wherein the flow rate may be, forexample, consistent and constant during steady-state operation.Moreover, the negative charge-based purification module includes asuspension of anionic resin beads, wherein the resin bead surfacecomprises anionic functionality configured to selectively associate withsaid biological product at a specific pH and ionic strength.

The negative charge-based purification module includes a lid systemcapable of motion along the z-axis having at least one gasketed lid, theat least one gasketed lid comprising at least one inlet to introduce agas to enable control of positive head pressure, at least one vent portto enable equilibration to atmospheric pressure, at least one inlet tointroduce a suspension of resin beads, at least one inlet to receive thefiltrate containing a biological product to enable association, at leasttwo inlets to introduce a buffer system to disperse the resin beads toenable washing of, dissociation from, or regeneration of said resinbeads; a mechanical rotary system capable of motion in the xy-plane, forexample, a carousel comprising at least two vessels charged with resinbeads that are configured to continuously receive a mixture containing abiological product and subsequently transport the resultingheterogeneous mixture containing a biological product, resin beads, abuffer, or any combination thereof; a collection system capable ofmotion along the z-axis that interfaces with at least one of the atleast two vessels of the mechanical rotary system to enable collectionof waste, the fraction containing the biological product, or anycombination thereof; at least one gas; at least one association/washbuffer system; at least one dissociation buffer system; at least oneresin bead regeneration buffer system; at least one collection vessel;at least one sensor or detector; and, at least one fluid handling pump.

The equipment design for the negative charge-based purification module(FIGS. 22-23) enables for automated and continuous, biologicalpurification as compared to a batch process or a semi-continuous processand provides for a small footprint.

As described herein, the negative charge-based purification moduleincludes a suspension of resin beads, in which the concentration dependson the desired charge or electrostatic association capacity or thedesired solution viscosity. Alternatively, the negative charge-basedpurification module resin bead size is micron to sub-micron and isdependent on the charge or electrostatic association capacity needs,which is a function of the surface area-to-volume ratio required toenable adequate fluid dynamics for equilibration and charge orelectrostatic interactions, for example, solution viscosity dependencyand surface charge density dependency, respectively.

The vessel number and size depends on input flow rate, resin bead chargeor electrostatic association capacity, and association equilibrationtime. The vessel material is selected to limit protein binding.

The filter or filter membrane material and pore size of the vesseldepends on the resin bead diameter and the biological product ofinterest. The filter or filter membrane materials is selected to limitprotein binding.

The material selection for the resin beads of the negative charge-basedpurification module is important for negligible leachables and toprovide for robust stability to enable recycling and reuse. The resinbeads may be solid, porous, nanoporous, microporous, or any combinationthereof.

Within the negative charge-based purification module, the anionicsurface selection is an important consideration and may include anionicpolymers, net negatively charged peptides or proteins, oligonucleotides,carboxyl functionality. Further, the selection is based on achievingappropriate electrostatic interactions and stability between thenegatively charged bead surface and the biological product within adefined buffer (pH and ionic strength).

The association/wash buffer of the negative charge-based purificationmodule is dependent on the biological product of interest (e.g., amonoclonal antibody) and small impurities to be removed. The pH, ionicstrength, use of surfactants, and use of organic and/or inorganic saltsis also contemplated in the association/wash buffer. Moreover,additional vessels and replicate carousel positions may also be requiredto effectively wash.

The dissociation buffer of the negative charge-based purification moduleis dependent on the strength of the electrostatic interactions betweenthe anionic resin bead surface functionality and the biological product(e.g., monoclonal antibody) of interest. For example, the dissociationbuffer may vary pH, ionic strength, use of surfactants, use of organicand/or inorganic salts, use of multiple dissociation buffer compositions(e.g., to increase yield, and which might require additional vessels andreplicate carousel positions). In other examples, multiple dissociationbuffers varying pH, ionic strength, or any combination thereof, areutilized sequentially to create a gradient dissociation effect.Moreover, additional vessels and replicate carousel positions oradditional dissociations may also be required to effectively dissociate.

The dwell time for each stage in the progression of the vessel throughthe rotary process is dependent on flow rate, equilibration times, andthroughput volume. Moreover, depending on buffer composition and pH,viral inactivation and removal during the wash steps is alsocontemplated, and would thus eliminate the need for a separate viralinactivation and removal process step, for example, when purifying amonoclonal antibody.

The negative charge-based purification module may include in-linesampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, optical density measurementdevices, UV detectors, RI detectors) may be used to enable feedbackcontrol mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the negativecharge-based, magnetic purification module utilizes a mobile affinityresin capable of in situ regeneration and recycling to enable moreefficient use of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Affinity-Based Purification Module Having a Staged Linear System

Provided herein is an affinity-based purification module for separatinga mixture into two or more fractions, at least one fraction containing abiological product. The module includes at least one inlet and at leastone outlet configured to permit continuous fluid flow between the atleast one inlet and the at least one outlet and wherein the flow ratemay be, for example, consistent and constant during steady-stateoperation. Moreover, the affinity-based purification module includes asuspension of affinity resin beads, wherein the resin bead surface,without intent to be limiting, is coupled to Protein A, Protein G,Protein L, an antigenic protein, a protein, a receptor, an antibody, oran aptamer configured to selectively bind said biological product.

The affinity-based purification module includes at least one gasketedlid system having at least one inlet to introduce a gas to enablecontrol of positive head pressure, at least one vent port to enableequilibration to atmospheric pressure, at least one inlet to introduce asuspension of resin beads, at least one inlet to receive the filtratecontaining a biological product to enable binding, at least two inletsto introduce a buffer system to disperse the resin beads to enablewashing of, elution from, or regeneration of said resin beads; a stagedlinear system comprising at least two vessels charged with mobile resinbeads that are configured to continuously receive a mixture containing abiological product and subsequently process the resulting heterogeneousmixture containing a biological product, resin beads, a buffer, or anycombination thereof; a collection system connected to at least one ofthe at least two vessels of the staged linear system to enablecollection of waste, the fraction containing the biological product, orany combination thereof; at least one gas; at least one binding/washbuffer system; at least one elution buffer system; at least one resinbead regeneration buffer system; at least one collection vessel; atleast one sensor or detector; and, at least one fluid handling pump.

The equipment design of the affinity-based purification module (FIGS.24A and 24B) enables for automated and continuous, biologicalpurification, as compared to a batch process or a semi-continuousprocess and provides for a small footprint.

As described herein, the affinity-based purification module includes asuspension of resin beads, in which the concentration depends on thedesired binding capacity or the desired solution viscosity.Alternatively, the affinity-based purification module resin bead size ismicron to sub-micron and is dependent on the binding capacity needs,which is a function of the surface area-to-volume ratio required toenable adequate fluid dynamics for equilibration and affinityinteractions, for example, solution viscosity dependency and surfaceligand density dependency, respectively.

The vessel number and size depends on input flow rate, resin beadbinding capacity, and binding equilibration time. The vessel material isselected to limit protein binding.

The filter or filter membrane material and pore size of the vesseldepends on the resin bead diameter and the biological product ofinterest. The filter or filter membrane materials is selected to limitprotein binding.

The material selection for the resin beads of the affinity-basedpurification module is important for negligible leachables and toprovide for robust stability to enable recycling and reuse. The resinbeads may be solid, porous, nanoporous, microporous, or any combinationthereof.

The binding/wash buffer is dependent on the biological product (e.g.,monoclonal antibody) of interest and small impurities to be removed.Other considerations for the binding/wash buffer including pH, ionicstrength, use of surfactants, and use of organic and/or inorganic saltsare also contemplated in the binding/wash buffer. Moreover, additionalvessels and replicate carousel positions may also be required toeffectively wash.

The elution buffer is dependent on the binding affinity (e.g., strengthof the non-covalent interactions) between the resin bead surface ligandsand the biological product (e.g., monoclonal antibody) of interest. Forexample, the elution buffer may vary pH, ionic strength, use ofsurfactants, use of organic and/or inorganic salts, use of multipleelution buffer compositions (e.g., to increase yield, and which mightrequire additional vessels and replicate carousel positions). Moreover,additional elutions may also be required to effectively elute.

Depending on buffer composition and pH, viral inactivation and removalduring the wash steps is also contemplated, and would thus eliminate theneed for a separate viral inactivation and removal process step, forexample, when purifying a monoclonal antibody.

The affinity-based, purification module may include in-line samplingports for in-process analytical testing and/or in-line analyticalmeasurement techniques. Further, the in-line analytical measurementtechniques (e.g., flow sensors, optical density measurement devices, UVdetectors, RI detectors) may be used to enable feedback controlmechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the affinity-basedpurification module utilizes a mobile affinity resin capable of in situregeneration and recycling to enable more efficient use of the resin andto enable continuous processing in a small-footprint without concerns oftraditional column capacity limitations.

Positive Charge-Based Purification Module Having a Staged Linear System

Provided herein is a positive charge-based purification module forseparating a mixture into two or more fractions, at least one fractioncontaining a biological product. The module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation. Moreover, the positive charge-based purificationmodule includes a suspension of affinity resin beads, wherein the resinbead surface, without intent to be limiting, is coupled to Protein A,Protein G, Protein L, an antigenic protein, a protein, a receptor, anantibody, or an aptamer configured to selectively bind said biologicalproduct.

The positive charge-based purification module includes at least onegasketed lid system having at least one inlet to introduce a gas toenable control of positive head pressure, at least one vent port toenable equilibration to atmospheric pressure, at least one inlet tointroduce a suspension of resin beads, at least one inlet to receive thefiltrate containing a biological product, at least two inlets tointroduce a buffer system to disperse the resin beads to enable washingof, dissociation from, or regeneration of said resin beads; a stagedlinear system comprising at least two vessels charged with mobile resinbeads that are configured to continuously receive a mixture containing abiological product and subsequently process the resulting heterogeneousmixture containing a biological product, resin beads, a buffer, or anycombination thereof; a collection system connected to at least one ofthe at least two vessels of the staged linear system to enablecollection of waste, the fraction containing the biological product, orany combination thereof; at least one gas; at least one binding/washbuffer system; at least one elution buffer system; at least one resinbead regeneration buffer system; at least one collection vessel; atleast one sensor or detector; and, at least one fluid handling pump.

The equipment design of the positive charge-based purification module(FIGS. 24A and 24B) enables for automated and continuous, biologicalpurification, as compared to a batch process or a semi-continuousprocess and provides for a small footprint.

As described herein, the positive charge-based purification moduleincludes cationic resin beads, in which the concentration depends on thedesired charge or electrostatic association capacity or the desiredsolution viscosity. Alternatively, the positive charge-basedpurification module resin bead size is micron to sub-micron and isdependent on the charge or electrostatic association capacity needs,which is a function of the surface area-to-volume ratio required toenable adequate fluid dynamics for equilibration and charge orelectrostatic interactions, for example, solution viscosity dependencyand surface charge density dependency, respectively.

The vessel number and size depends on input flow rate, resin bead chargeor electrostatic association capacity, and association equilibrationtime. The vessel material is selected to limit protein binding.

The filter or filter membrane material and pore size of the vesseldepends on the resin bead diameter and the biological product ofinterest. The filter or filter membrane materials is selected to limitprotein binding.

The material selection for the resin beads of the positive charge-basedpurification module is important for negligible leachables and toprovide for robust stability to enable recycling and reuse. The resinbeads may be solid, porous, nanoporous, microporous, or any combinationthereof.

Within the positive charge-based purification module, the cationicsurface selection is an important consideration and may include cationicpolymers, net positively charged peptides or proteins, aminefunctionality. Further, the selection is based on achieving appropriateelectrostatic interactions and stability between the positively chargedbead surface and the biological product within a defined buffer (pH andionic strength).

The association/wash buffer of the positive charge-based purificationmodule is dependent on the biological product of interest (e.g., amonoclonal antibody) and small impurities to be removed. The pH, ionicstrength, use of surfactants, and use of organic and/or inorganic saltsis also contemplated in the association/wash buffer. Moreover,additional vessels and replicate carousel positions may also be requiredto effectively wash.

The dissociation buffer of the positive charge-based purification moduleis dependent on the strength of the electrostatic interactions betweenthe anionic resin bead surface functionality and the biological product(e.g., monoclonal antibody) of interest. For example, the dissociationbuffer may vary pH, ionic strength, use of surfactants, use of organicand/or inorganic salts, use of multiple dissociation buffer compositions(e.g., to increase yield, and which might require additional vessels andreplicate carousel positions). In other examples, multiple dissociationbuffers varying pH, ionic strength, or any combination thereof, areutilized sequentially to create a gradient dissociation effect.Moreover, additional dissociations may also be required to effectivelydissociate.

Depending on buffer composition and pH, viral inactivation and removalduring the wash steps is also contemplated, and would thus eliminate theneed for a separate viral inactivation and removal process step, forexample, when purifying a monoclonal antibody.

The positive charge-based purification module may include in-linesampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, optical density measurementdevices, UV detectors, RI detectors) may be used to enable feedbackcontrol mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the positivecharge-based purification module utilizes a mobile affinity resincapable of in situ regeneration and recycling to enable more efficientuse of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Negative Charge-Based Purification Module Having a Staged Linear System

Provided herein is a negative charge-based purification module forseparating a mixture into two or more fractions, at least one fractioncontaining a biological product. The module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation. Moreover, the negative charge-based purificationmodule includes a suspension of affinity resin beads, wherein the resinbead surface, without intent to be limiting, is coupled to Protein A,Protein G, Protein L, an antigenic protein, a protein, a receptor, anantibody, or an aptamer configured to selectively bind said biologicalproduct.

The negative charge-based purification module includes at least onegasketed lid system having at least one inlet to introduce a gas toenable control of positive head pressure, at least one vent port toenable equilibration to atmospheric pressure, at least one inlet tointroduce a suspension of resin beads, at least one inlet to receive thefiltrate containing a biological product, at least two inlets tointroduce a buffer system to disperse the resin beads to enable washingof, dissociation from, or regeneration of said resin beads; a stagedlinear system comprising at least two vessels charged with mobile resinbeads that are configured to continuously receive a mixture containing abiological product and subsequently process the resulting heterogeneousmixture containing a biological product, resin beads, a buffer, or anycombination thereof; a collection system connected to at least one ofthe at least two vessels of the staged linear system to enablecollection of waste, the fraction containing the biological product, orany combination thereof; at least one gas; at least one binding/washbuffer system; at least one elution buffer system; at least one resinbead regeneration buffer system; at least one collection vessel; atleast one sensor or detector; and, at least one fluid handling pump.

The equipment design of the negative charge-based purification module(FIGS. 25A and 25B) enables for automated and continuous, biologicalpurification, as compared to a batch process or a semi-continuousprocess and provides for a small footprint.

As described herein, the negative charge-based purification moduleincludes a suspension of resin beads, in which the concentration dependson the desired charge or electrostatic association capacity or thedesired solution viscosity. Alternatively, the negative charge-basedpurification module resin bead size is micron to sub-micron and isdependent on the charge or electrostatic association capacity needs,which is a function of the surface area-to-volume ratio required toenable adequate fluid dynamics for equilibration and charge orelectrostatic interactions, for example, solution viscosity dependencyand surface charge density dependency, respectively.

The vessel number and size depends on input flow rate, resin bead chargeor electrostatic association capacity, and association equilibrationtime. The vessel material is selected to limit protein binding.

The filter or filter membrane material and pore size of the vesseldepends on the resin bead diameter and the biological product ofinterest. The filter or filter membrane materials is selected to limitprotein binding.

The material selection for the resin beads of the negative charge-basedpurification module is important for negligible leachables and toprovide for robust stability to enable recycling and reuse. The resinbeads may be solid, porous, nanoporous, microporous, or any combinationthereof.

Within the negative charge-based purification module, the anionicsurface selection is an important consideration and may include anionicpolymers, net negatively charged peptides or proteins, oligonucleotides,carboxyl functionality. Further, the selection is based on achievingappropriate electrostatic interactions and stability between thenegatively charged bead surface and the biological product within adefined buffer (pH and ionic strength).

The association/wash buffer of the negative charge-based purificationmodule is dependent on the biological product of interest (e.g., amonoclonal antibody) and small impurities to be removed. The pH, ionicstrength, use of surfactants, and use of organic and/or inorganic saltsis also contemplated in the association/wash buffer. Moreover,additional vessels and replicate carousel positions may also be requiredto effectively wash.

The dissociation buffer of the negative charge-based purification moduleis dependent on the strength of the electrostatic interactions betweenthe anionic resin bead surface functionality and the biological product(e.g., monoclonal antibody) of interest. For example, the dissociationbuffer may vary pH, ionic strength, use of surfactants, use of organicand/or inorganic salts, use of multiple dissociation buffer compositions(e.g., to increase yield, and which might require additional vessels andreplicate carousel positions). In other examples, multiple dissociationbuffers varying pH, ionic strength, or any combination thereof, areutilized sequentially to create a gradient dissociation effect.Moreover, additional dissociations may also be required to effectivelydissociate.

Depending on buffer composition and pH, viral inactivation and removalduring the wash steps is also contemplated, and would thus eliminate theneed for a separate viral inactivation and removal process step, forexample, when purifying a monoclonal antibody.

The negative charge-based purification module may include in-linesampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, optical density measurementdevices, UV detectors, RI detectors) may be used to enable feedbackcontrol mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the negativecharge-based, magnetic purification module utilizes a mobile affinityresin capable of in situ regeneration and recycling to enable moreefficient use of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Affinity-Based, Fluidic Purification Module

As provided herein, an affinity-based, fluidic purification module forseparating a mixture into two or more fractions, at least one fractioncontaining a biological product is described. The affinity-based,fluidic purification module includes at least one inlet and at least oneoutlet configured to permit continuous fluid flow between the at leastone inlet and the at least one outlet and wherein the flow rate may be,for example, consistent and constant during steady-state operation.Moreover, the affinity-based, fluidic purification module includes asuspension of affinity magnetic resin beads, wherein the magnetic resinbead surface, without intent to be limiting, is coupled to Protein A,Protein G, Protein L, an antigenic protein, a protein, a receptor, anantibody, or an aptamer configured to selectively bind said biologicalproduct.

The affinity-based, fluidic purification module includes at least oneequilibration vessel to allow for binding of the biological product tothe magnetic resin bead surface; at least one low pH equilibrationvessel to allow for de-binding of the biological product from themagnetic resin bead surface; at least one first hybrid cross-flowfluidic device or chip (e.g., a microfluidic, a mesofluidic, amillifluidic, a macrofluidic device or chip, or any combination thereof)comprising at least one magnetic field and at least one of apiezoelectric component or a dielectrophoretic electrode configured togenerate or induce a unidirectional force to manipulate the flow path ofthe magnetic resin beads in said heterogeneous mixture, and a cross-flowchannel to separate the magnetic resin beads bound to the biologicalproduct from small impurities in the heterogeneous mixture (FIG. 28); atleast one second hybrid cross-flow fluidic device or chip (e.g., amicrofluidic, a mesofluidic, a millifluidic, a macrofluidic device orchip, or any combination thereof) comprising at least one magnetic fieldand at least one of a piezoelectric component or a dielectrophoreticelectrode configured to generate or induce a unidirectional force tomanipulate the flow path of said magnetic resin beads from saidbiological product, and a cross-flow channel to separate the magneticresin beads from said biological product (FIG. 28); at least two buffersystems; at least one magnetic resin bead regeneration buffer system; atleast one regeneration equilibration vessel configured to enablerecycling of said magnetic resin beads; at least one collection vessel;at least one sensor or detector; and, at least one fluid handling pump.

The equipment design for the affinity-based, fluidic purification module(FIG. 29) enables continuous, biological magnetic purification andprovides for a small footprint.

The at least one equilibration vessel volume and agitation capabilitiesof the affinity-based, fluidic purification module consider the inputflow rate and throughput, equilibration time and agitation rate toenable binding kinetics, and magnetic resin bead concentration andbinding capacity. The equilibration buffer for the affinity-based,fluidic purification module is dependent on the biological product ofinterest (e.g., a monoclonal antibody) and the small impurities to beremoved. Considerations for the buffer include pH, ionic strength, useof surfactants, or use of organic and/or inorganic salts.

The cross-flow channel size of the affinity-based, fluidic purificationmodule is dependent on the solution viscosity, magnetic resin beadconcentration, input flow rate and throughput volume. The piezoelectricor acoustic actuator considers the physical location and the energy(e.g., frequency) to enable desired magnetic resin bead deflection ormanipulation of the magnetic resin bead flow path, and the piezoelectriccrystal type. The dielectrophoretic electrodes consider selective-typeand design to enable desired magnetic resin bead deflection, the numberof electrodes and spacing to enable desired bead deflection, the voltageapplied to enable desired bead deflection, and the electrode material.

The magnetic field strength and field effect are dependent on the flowrate and magnetic resin bead concentration, size, saturationmagnetization, and magnetic susceptibility, and/or the proximity to thecross-flow channel. In embodiments, the magnetic field strength rangesfrom about 0.01 Tesla to about 1 Tesla (e.g., up to about 1 Tesla). Themagnetic resin bead concentration is dependent on the desired bindingcapacity, desired solution viscosity, and the magnetic resin bead sizeis dependent on the surface area-to-volume ratio to enable adequatefluid dynamics for equilibration and affinity interactions, for example,solution viscosity dependency and surface ligand density dependency,respectively. In embodiments, the magnetic resin beads have sub-micronto micron diameters.

The material selection of the affinity-based, fluidic purificationmodule is important for negligible leachables and robust stability toenable recycling and reuse of the affinity-based, fluidic purificationmodule. The magnetic resin beads may be solid, porous, nanoporous,microporous, or any combination thereof.

The at least one low pH equilibration vessel volume and agitationcapabilities of the affinity-based, fluidic purification module considerthe input flow rate and throughput, equilibration time to enablede-binding kinetics, a low pH elution buffer at 10× to enable dilutionduring equilibration time to arrive at 1× final buffer saltconcentration. The low pH elution buffer is dependent on the bindingaffinity of biological product of interest (e.g., a monoclonal antibody)for the magnetic resin bead surface ligand, and variations may includepH, ionic strength, or use of organic and/or inorganic salts.

The throughput of the affinity-based, fluidic purification module may beincreased by multiplexing multiple fluidic devices or chips, in seriesor in parallel. Moreover, multiple fluidic devices or chips in series orin parallel may be required to enable complete purification.Additionally, a regeneration equilibration vessel might require apermanent magnetic field and a waste line to maintain correctconcentration of magnetic resin beads and allow for effective recycling.Viral inactivation and removal may be accomplished during the low pHelution buffer equilibration and subsequent fluidic processing stepsdepending on buffer composition and pH.

The affinity-based, fluidic purification module may include at least onetangential flow filtration system operated in fed-batch or perfusionmode to concentrate and buffer exchange the fraction containing thebiological product.

The affinity-based, fluidic purification module may include in-linesampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, pressure sensors, opticaldensity measurement devices, UV detectors, RI detectors) may be used toenable feedback control mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the affinity-based,fluidic purification module utilizes a mobile affinity resin capable ofin situ regeneration and recycling to enable more efficient use of theresin and to enable continuous processing in a small-footprint withoutconcerns of traditional column capacity limitations.

Positive Charge-Based, Fluidic Purification Module

As provided herein, a positive charge-based, fluidic purification modulefor separating a mixture into two or more fractions, at least onefraction containing a biological product is described. The positivecharge-based, fluidic purification module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation. Moreover, the positive charge-based, fluidicpurification module includes a suspension of cationic magnetic resinbeads, wherein the magnetic resin bead surface comprises cationicfunctionality configured to selectively associate with said biologicalproduct based on charge or electrostatic interactions at a specific pHand ionic strength.

The positive charge-based, fluidic purification module includes at leastone association equilibration vessel to allow for charge orelectrostatic association of the biological product with the magneticresin bead surface; at least one dissociation equilibration vessel toallow for dissociation of the biological product from the magnetic resinbead surface; at least one first hybrid cross-flow fluidic device orchip (e.g., a microfluidic, a mesofluidic, a millifluidic, amacrofluidic device or chip, or any combination thereof) comprising atleast one magnetic field and at least one of a piezoelectric componentor a dielectrophoretic electrode configured to generate or induce aunidirectional force to manipulate the flow path of the magnetic resinbeads in said heterogeneous mixture, and a cross-flow channel toseparate the magnetic resin beads bound to the biological product fromsmall impurities in the heterogeneous mixture (FIG. 28); at least onesecond hybrid cross-flow fluidic device or chip (e.g., a microfluidic, amesofluidic, a millifluidic, a macrofluidic device or chip, or anycombination thereof) comprising at least one magnetic field and at leastone of a piezoelectric component or a dielectrophoretic electrodeconfigured to generate or induce a unidirectional force to manipulatethe flow path of said magnetic resin beads from said biological product,and a cross-flow channel to separate the magnetic resin beads from saidbiological product (FIG. 28); at least two buffer systems; at least onemagnetic resin bead regeneration buffer system; at least oneregeneration equilibration vessel configured to enable recycling of saidmagnetic resin beads; at least one collection vessel; at least onesensor or detector; and, at least one fluid handling pump.

The equipment design for the positive charge-based, fluidic purificationmodule (FIG. 30) enables continuous, biological magnetic purificationand provides for a small footprint.

The at least one association equilibration vessel volume and agitationcapabilities of the positive charge-based, fluidic purification moduleconsider the input flow rate and throughput, equilibration time andagitation rate to enable association kinetics, and magnetic resin beadconcentration and charge or electrostatic association capacity. Theassociation buffer for the positive charge-based, fluidic purificationmodule is dependent on the biological product of interest (e.g., amonoclonal antibody) and the small impurities to be removed.Considerations for the buffer include pH, ionic strength, use ofsurfactants, or use of organic and/or inorganic salts, specifically tomaintain favorable charge or electrostatic interactions between thetarget monoclonal antibody and positively charged bead surface.

The cross-flow channel size of the positive charge-based, fluidicpurification module is dependent on the solution viscosity and magneticresin bead concentration, and input flow rate and throughput volume. Thepiezoelectric or acoustic actuator considers the physical location andthe energy (e.g., frequency) to enable desired magnetic resin beaddeflection or manipulation of the magnetic resin bead flow path, and thepiezoelectric crystal type. The dielectrophoretic electrodes considerselective-type and design to enable desired magnetic resin beaddeflection, the number of electrodes and spacing to enable desired beaddeflection, the voltage applied to enable desired bead deflection, andthe electrode material.

The magnetic field strength and field effect are dependent on the flowrate and magnetic resin bead concentration, size, saturationmagnetization, and magnetic susceptibility, and/or the proximity to thecross-flow channel. In embodiments, the magnetic field strength rangesfrom about 0.01 Tesla to about 1 Tesla (e.g., up to about 1 Tesla). Themagnetic resin bead concentration is dependent on the desired charge orelectrostatic association capacity, desired solution viscosity, and themagnetic resin bead size is dependent on the surface area-to-volumeratio required to enable adequate fluid dynamics for equilibration andcharge or electrostatic interactions, for example, solution viscositydependency and surface charge density dependency, respectively. Inembodiments, the magnetic resin beads have sub-micron to microndiameters.

The material selection of the positive charge-based, fluidicpurification module is important for negligible leachables and robuststability to enable recycling and reuse in the positive charge-based,fluidic purification module.

The cationic surface selection for the positive charge-based, fluidicpurification module is important and may include cationic polymers, netpositively charged peptides or proteins, amine functionality, andselection is based on achieving appropriate charge or electrostaticinteractions and association stability between the positively chargedbead surface and the biological product within a defined buffer (pH andionic strength).

The at least one dissociation equilibration vessel volume and agitationcapabilities of the positive charge-based, fluidic purification moduleconsider the input flow rate and throughput, equilibration time toenable dissociation kinetics, a dissociation buffer at 10× to enabledilution during equilibration time to arrive at 1× final buffer saltconcentration. The dissociation buffer is dependent on the strength ofthe charge or electrostatic interactions between the biological productof interest (e.g., a monoclonal antibody) and the magnetic resin beadcationic surface, and variations may include pH, ionic strength, use ofsurfactants, or use of organic and/or inorganic salts. In embodiments,multiple dissociation equilibration vessels comprising discrete buffersvarying pH, ionic strength, or any combination thereof, are utilizedsequentially to create a gradient dissociation effect.

The throughput of the positive charge-based, fluidic purification modulemay be increased by multiplexing multiple fluidic devices or chips, inseries or in parallel. Moreover, multiple fluidic devices or chips inseries or in parallel may be required to enable complete purification.Additionally, a regeneration equilibration vessel might require apermanent magnetic and a waste line to maintain correct concentration ofmagnetic resin beads and allow for effective recycling. Viralinactivation and removal may be accomplished during the association ordissociation buffer equilibration and subsequent fluidic processingsteps depending on buffer composition and pH.

The positive charge-based, fluidic purification module may include atleast one tangential flow filtration system operated in fed-batch orperfusion mode to concentrate and buffer exchange the fractioncontaining the biological product.

The positive charge-based, fluidic purification module may includein-line sampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, pressure sensors, opticaldensity measurement devices, UV detectors, RI detectors) may be used toenable feedback control mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the positivecharge-based, fluidic purification module utilizes a mobile affinityresin capable of in situ regeneration and recycling to enable moreefficient use of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Negative Charge-Based, Fluidic Purification Module

As provided herein, a negative charge-based, fluidic purification modulefor separating a mixture into two or more fractions, at least onefraction containing a biological product is described. The negativecharge-based, fluidic purification module includes at least one inletand at least one outlet configured to permit continuous fluid flowbetween the at least one inlet and the at least one outlet and whereinthe flow rate may be, for example, consistent and constant duringsteady-state operation. Moreover, the negative charge-based, fluidicpurification module includes a suspension of anionic magnetic resinbeads, wherein the magnetic resin bead surface comprises anionicfunctionality configured to selectively associate with said biologicalproduct based on charge or electrostatic interactions at a specific pHand ionic strength.

The negative charge-based, fluidic purification module includes at leastone association equilibration vessel to allow for charge orelectrostatic association of the biological product with the magneticresin bead surface; at least one dissociation equilibration vessel toallow for dissociation of the biological product from the magnetic resinbead surface; at least one first hybrid cross-flow fluidic device orchip (e.g., a microfluidic, a mesofluidic, a millifluidic, amacrofluidic device or chip, or any combination thereof) comprising atleast one magnetic field and at least one of a piezoelectric componentor a dielectrophoretic electrode configured to generate or induce aunidirectional force to manipulate the flow path of the magnetic resinbeads in said heterogeneous mixture, and a cross-flow channel toseparate the magnetic resin beads bound to the biological product fromsmall impurities in the heterogeneous mixture (FIG. 28); at least onesecond hybrid cross-flow fluidic device or chip (e.g., a microfluidic, amesofluidic, a millifluidic, a macrofluidic device or chip, or anycombination thereof) comprising at least one magnetic field and at leastone of a piezoelectric component or a dielectrophoretic electrodeconfigured to generate or induce a unidirectional force to manipulatethe flow path of said magnetic resin beads from said biological product,and a cross-flow channel to separate the magnetic resin beads from saidbiological product (FIG. 28); at least two buffer systems; at least onemagnetic resin bead regeneration buffer system; at least oneregeneration equilibration vessel configured to enable recycling of saidmagnetic resin beads; at least one collection vessel; at least onesensor or detector; and, at least one fluid handling pump.

The equipment design for the negative charge-based, fluidic purificationmodule (FIG. 30) enables continuous, biological magnetic purificationand provides for a small footprint.

The at least one association equilibration vessel volume and agitationcapabilities of the negative charge-based, fluidic purification moduleconsider the input flow rate and throughput, equilibration time andagitation rate to enable association kinetics, and magnetic resin beadconcentration and charge or electrostatic association capacity. Thebuffer for the negative charge-based, fluidic purification module isdependent on the biological product of interest (e.g., a monoclonalantibody) and the small impurities to be removed. Considerations for thebuffer include pH, ionic strength, use of surfactants, or use of organicand/or inorganic salts, specifically to maintain favorable charge orelectrostatic interactions between the target monoclonal antibody andnegatively charged bead surface.

The cross-flow channel size of the negative charge-based, fluidicpurification module is dependent on the solution viscosity and magneticresin bead concentration, and input flow rate and throughput volume. Thepiezoelectric or acoustic actuator considers the physical location andthe energy (e.g., frequency) to enable desired magnetic resin beaddeflection or manipulation of the magnetic resin bead flow path, and thepiezoelectric crystal type. The dielectrophoretic electrodes considerselective-type and design to enable desired magnetic resin beaddeflection, the number of electrodes and spacing to enable desired beaddeflection, the voltage applied to enable desired bead deflection, andthe electrode material.

The magnetic field strength and field effect are dependent on the flowrate and magnetic resin bead concentration, size, saturationmagnetization, and magnetic susceptibility, and/or the proximity to thecross-flow channel. In embodiments, the magnetic field strength rangesfrom about 0.01 Tesla to about 1 Tesla (e.g., up to about 1 Tesla). Themagnetic resin bead concentration is dependent on the desired charge orelectrostatic association capacity, desired solution viscosity, and themagnetic resin bead size is dependent on the surface area-to-volumeratio required to enable adequate fluid dynamics for equilibration andcharge or electrostatic interactions, for example, solution viscositydependency and surface charge density dependency, respectively. Inembodiments, the magnetic resin beads have sub-micron to microndiameters.

The material selection of the negative charge-based, fluidicpurification module is important for negligible leachables and robuststability to enable recycling and reuse in the negative charge-based,fluidic purification module.

The anionic surface selection for the negative charge-based, fluidicpurification module is important and may include anionic polymers, netnegatively charged peptides or proteins, carboxyl functionality, andselection is based on achieving appropriate charge or electrostaticinteractions and association stability between the negatively chargedbead surface and the biological product within a defined buffer (pH andionic strength).

The at least one dissociation equilibration vessel volume and agitationcapabilities of the negative charge-based, fluidic purification moduleconsider the input flow rate and throughput, equilibration time toenable dissociation kinetics, a dissociation buffer at 10× to enabledilution during equilibration time to arrive at 1× final buffer saltconcentration. The dissociation buffer is dependent on the strength ofthe charge or electrostatic interactions between the biological productof interest (e.g., a monoclonal antibody) and the magnetic resin beadanionic surface, and variations may include pH, ionic strength, use ofsurfactants, or use of organic and/or inorganic salts. In embodiments,multiple dissociation equilibration vessels comprising discrete buffersvarying pH, ionic strength, or any combination thereof, are utilizedsequentially to create a gradient dissociation effect.

The throughput of the negative charge-based, fluidic purification modulemay be increased by multiplexing multiple fluidic devices or chips, inseries or in parallel. Moreover, multiple fluidic devices or chips inseries or in parallel may be required to enable complete purification.Additionally, a regeneration equilibration vessel might require apermanent magnetic and a waste line to maintain correct concentration ofmagnetic resin beads and allow for effective recycling. Viralinactivation and removal may be accomplished during the association ordissociation buffer equilibration and subsequent fluidic processingsteps depending on buffer composition and pH.

The negative charge-based, fluidic purification module may include atleast one tangential flow filtration system operated in fed-batch orperfusion mode to concentrate and buffer exchange the fractioncontaining the biological product.

The negative charge-based, fluidic purification module may includein-line sampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, pressure sensors, opticaldensity measurement devices, UV detectors, RI detectors) may be used toenable feedback control mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the negativecharge-based, magnetic purification module utilizes a mobile affinityresin capable of in situ regeneration and recycling to enable moreefficient use of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Affinity-Based TFF Purification Module

As provided herein, an affinity-based TFF purification module forseparating a mixture into two or more fractions, at least one fractioncontaining a biological product is described. The affinity-based TFFpurification module includes at least one inlet and at least one outletconfigured to permit continuous fluid flow between the at least oneinlet and the at least one outlet and wherein the flow rate isconsistent and constant during steady-state operation. Moreover, theaffinity-based TFF purification module includes a suspension of affinityresin beads, wherein the resin bead surface, without intent to belimiting, is coupled to Protein A, Protein G, Protein L, an antigenicprotein, a protein, a receptor, an antibody, or an aptamer configured toselectively bind said biological product.

The affinity-based TFF purification module includes at least oneequilibration vessel to allow for binding of the biological product tothe resin bead surface and at least one first tangential flow filtrationsystem comprising a hollow fiber membrane filter to separate the resinbeads bound to the biological product from small impurities in theheterogeneous mixture; at least one low pH equilibration vessel to allowfor de-binding of the biological product from the resin bead surface andat least one second tangential flow filtration system comprising ahollow fiber membrane filter to separate the resin beads from saidunbound biological product; at least one regeneration equilibrationvessel and at least one third tangential flow filtration systemcomprising a hollow fiber membrane filter to concentrate and bufferexchange the resin beads to enable their recycling and reuse; at leastone collection vessel and at least one fourth tangential flow filtrationsystem to allow for concentration and buffer exchange of the biologicalproduct; at least two buffer systems; at least one resin bead recyclingbuffer system; at least one sensor or detector; and, at least one fluidhandling pump.

The equipment design for the affinity-based TFF purification module(FIG. 31) enables continuous, biological purification and provides for asmall footprint.

The at least one equilibration vessel volume and agitation capabilitiesof the affinity-based TFF purification module consider the input flowrate and throughput, equilibration time and agitation rate to enablebinding kinetics, and resin bead concentration and binding capacity. Theequilibration buffer for the affinity-based TFF purification module isdependent on the biological product of interest (e.g., a monoclonalantibody) and the small impurities to be removed. Considerations for thebuffer include pH, ionic strength, use of surfactants, or use of organicand/or inorganic salts.

The hollow fiber membrane filter length and surface area of theaffinity-based TFF purification module is dependent on the solutionviscosity, small impurities and resin bead concentration, and input flowrate and throughput volume. The hollow fiber membrane material isselected from low protein binding materials, including, but not limitedto, PES, mPES, or MCE. The pore size of the hollow fiber membrane isselected from the range of about 10 kDa to about 1 μm. The innerdiameter of the hollow fiber membrane is selected from the range ofabout 0.5 mm to about 5 mm.

The resin bead concentration is dependent on the desired bindingcapacity, desired solution viscosity, and the resin bead size isdependent on the surface area-to-volume ratio to enable adequate fluiddynamics for equilibration and affinity interactions, for example,solution viscosity dependency and surface ligand density dependency,respectively. In embodiments, the resin beads have micron diameters.

The resin bead selection of the affinity-based TFF purification moduleis important for negligible leachables and robust stability to enablerecycling and reuse of the affinity-based purification module.

The at least one low pH equilibration vessel volume and agitationcapabilities of the affinity-based TFF purification module consider theinput flow rate and throughput, equilibration time to enable de-bindingkinetics, and a low pH elution buffer, for example, a low pH elutionbuffer at 10× to enable dilution during equilibration time to arrive at1× final buffer salt concentration. The low pH elution buffer isdependent on the binding affinity of biological product of interest(e.g., a monoclonal antibody) for the resin bead surface ligand, andvariations may include pH, ionic strength, or use of organic and/orinorganic salts.

The at least one regeneration equilibration vessel of the affinity-basedTFF purification module is used in combination with the at least onethird tangential flow filtration system to allow for concentration andbuffer exchange of the resin beads to return the resin beads to theirinitial condition, thus, enabling recycling and reuse of the resinbeads.

The at least one collection vessel of the affinity-based TFFpurification module is used in combination with the at least one fourthtangential flow filtration system to allow for concentration and bufferexchange of the fraction containing the biological product.

In embodiments, the at least one equilibration vessel, the at least onelow pH equilibration vessel, and the at least one regenerationequilibration vessel of the affinity-based TFF purification module maycomprise a single vessel that is transitioned between the correspondingtangential flow filtration systems to enable purification andregeneration of the resin beads with appropriate buffers, whilemaintaining continuous flow of the filtrate via at least one additionalvessel on a parallel flow path.

In embodiments, the regeneration of the resin beads may be accomplishedwith the at least one low pH equilibration vessel and the at least onesecond tangential flow filtration system of the affinity-based TFFpurification module configured to comprise both the low pH elutionbuffer and the regeneration buffer to enable purification, concentrationand buffer exchange, thus regenerating the resin beads withoutnecessitating a separate regeneration equilibration vessel andcorresponding tangential flow filtration system.

Viral inactivation and/or removal may be accomplished during the low pHelution buffer equilibration and subsequent fluidic processing stepsdepending on buffer composition and pH.

The affinity-based TFF purification module may include in-line samplingports for in-process analytical testing and/or in-line analyticalmeasurement techniques. Further, the in-line analytical measurementtechniques (e.g., flow sensors, pressure sensors, optical densitymeasurement devices, UV detectors, RI detectors) may be used to enablefeedback control mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the affinity-based TFFpurification module utilizes a mobile affinity resin capable of in situregeneration and recycling to enable more efficient use of the resin andto enable continuous processing in a small-footprint without concerns oftraditional column capacity limitations.

Positive Charge-Based TFF Purification Module

As provided herein, a positive charge-based TFF purification module forseparating a mixture into two or more fractions, at least one fractioncontaining a biological product is described. The positive charge-basedTFF purification module includes at least one inlet and at least oneoutlet configured to permit continuous fluid flow between the at leastone inlet and the at least one outlet and wherein the flow rate isconsistent and constant during steady-state operation. Moreover, thepositive charge-based TFF purification module includes a suspension ofcationic resin beads, wherein the resin bead surface comprises cationicfunctionality configured to selectively associate with said biologicalproduct based on charge or electrostatic interactions at a specific pHand ionic strength.

The positive charge-based TFF purification module includes at least oneassociation equilibration vessel to allow for charge or electrostaticassociation of the biological product with the resin bead surface and atleast one first tangential flow filtration system comprising a hollowfiber membrane filter to separate the resin beads associated with thebiological product from small impurities in the heterogeneous mixture;at least one dissociation equilibration vessel to allow for dissociationof the biological product from the resin bead surface and at least onesecond tangential flow filtration system comprising a hollow fibermembrane filter to separate the resin beads from said dissociatedbiological product; at least one regeneration equilibration vessel andat least one third tangential flow filtration system comprising a hollowfiber membrane filter to concentrate and buffer exchange the resin beadsto enable their recycling and reuse; at least one collection vessel andat least one fourth tangential flow filtration system to allow forconcentration and buffer exchange of the biological product; at leasttwo buffer systems; at least one resin bead recycling buffer system; atleast one sensor or detector; and, at least one fluid handling pump.

The equipment design for the positive charge-based TFF purificationmodule (FIG. 32) enables continuous, biological purification andprovides for a small footprint.

The at least one association equilibration vessel volume and agitationcapabilities of the positive charge-based TFF purification moduleconsider the input flow rate and throughput, equilibration time andagitation rate to enable association kinetics, and resin beadconcentration and charge or electrostatic association capacity. Theassociation buffer for the positive charge-based TFF purification moduleis dependent on the biological product of interest (e.g., a monoclonalantibody) and the small impurities to be removed. Considerations for thebuffer include pH, ionic strength, use of surfactants, or use of organicand/or inorganic salts, specifically to maintain favorable charge orelectrostatic interactions between the target monoclonal antibody andpositively charged bead surface.

The hollow fiber membrane filter length and surface area of the positivecharge-based TFF purification module is dependent on the solutionviscosity, small impurities and resin bead concentration, and input flowrate and throughput volume. The hollow fiber membrane material isselected from low protein binding materials, including, but not limitedto, PES, mPES, or MCE. The pore size of the hollow fiber membrane isselected from the range of about 10 kDa to about 1 μm. The innerdiameter of the hollow fiber membrane is selected from the range ofabout 0.5 mm to about 5 mm.

The resin bead selection of the positive charge-based TFF purificationmodule is important for negligible leachables and robust stability toenable recycling and reuse in the positive charge-based TFF purificationmodule.

The cationic surface selection for the positive charge-based TFFpurification module is important and may include cationic polymers, netpositively charged peptides or proteins, amine functionality, andselection is based on achieving appropriate charge or electrostaticinteractions and association stability between the positively chargedbead surface and the biological product within a defined buffer (pH andionic strength).

The at least one dissociation equilibration vessel volume and agitationcapabilities of the positive charge-based TFF purification moduleconsider the input flow rate and throughput, equilibration time toenable dissociation kinetics, and a dissociation buffer, for example, adissociation buffer at 10× to enable dilution during equilibration timeto arrive at 1× final buffer salt concentration. The dissociation bufferis dependent on the strength of the charge or electrostatic interactionsbetween the biological product of interest (e.g., a monoclonal antibody)and the resin bead cationic surface, and variations may include pH,ionic strength, use of surfactants, or use of organic and/or inorganicsalts. In some aspects, multiple dissociation equilibration vessels areutilized with multiple tangential flow filtration systems to achieve agradient dissociation, for example, a pH gradient or an ionic strengthgradient.

The at least one regeneration equilibration vessel of the positivecharge-based TFF purification module is used in combination with the atleast one third tangential flow filtration system to allow forconcentration and buffer exchange of the resin beads to return the resinbeads to their initial condition, thus, enabling recycling and reuse ofthe resin beads.

The at least one collection vessel of the positive charge-based TFFpurification module is used in combination with the at least one fourthtangential flow filtration system to allow for concentration and bufferexchange of the fraction containing the biological product.

In embodiments, the at least one association equilibration vessel, theat least one dissociation equilibration vessel, and the at least oneregeneration equilibration vessel of the positive charge-based TFFpurification module may comprise a single vessel that is transitionedbetween the corresponding tangential flow filtration systems to enablepurification and regeneration of the resin beads with appropriatebuffers, while maintaining continuous flow of the filtrate via at leastone additional vessel on a parallel flow path.

In embodiments, the regeneration of the resin beads may be accomplishedwith solely the at least one dissociation equilibration vessel and theat least one second tangential flow filtration system of the positivecharge-based TFF purification module configured to comprise both the lowpH elution buffer and the regeneration buffer to enable purification,concentration and buffer exchange, thus regenerating the resin beadswithout necessitating a separate regeneration equilibration vessel andcorresponding tangential flow filtration system.

Viral inactivation and/or removal may be accomplished during theassociation or dissociation buffer equilibration and subsequent fluidicprocessing steps depending on buffer composition and pH.

The positive charge-based TFF purification module may include in-linesampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, pressure sensors, opticaldensity measurement devices, UV detectors, RI detectors) may be used toenable feedback control mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the positivecharge-based TFF purification module utilizes a mobile affinity resincapable of in situ regeneration and recycling to enable more efficientuse of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Negative Charge-Based TFF Purification Module

As provided herein, a negative charge-based TFF purification module forseparating a mixture into two or more fractions, at least one fractioncontaining a biological product is described. The negative charge-basedTFF purification module includes at least one inlet and at least oneoutlet configured to permit continuous fluid flow between the at leastone inlet and the at least one outlet and wherein the flow rate isconsistent and constant during steady-state operation. Moreover, thenegative charge-based TFF purification module includes a suspension ofanionic resin beads, wherein the resin bead surface comprises anionicfunctionality configured to selectively associate with said biologicalproduct based on charge or electrostatic interactions at a specific pHand ionic strength.

The negative charge-based TFF purification module includes at least oneassociation equilibration vessel to allow for charge or electrostaticassociation of the biological product with the resin bead surface and atleast one first tangential flow filtration system comprising a hollowfiber membrane filter to separate the resin beads associated with thebiological product from small impurities in the heterogeneous mixture;at least one dissociation equilibration vessel to allow for dissociationof the biological product from the resin bead surface and at least onesecond tangential flow filtration system comprising a hollow fibermembrane filter to separate the resin beads from said dissociatedbiological product; at least one regeneration equilibration vessel andat least one third tangential flow filtration system comprising a hollowfiber membrane filter to concentrate and buffer exchange the resin beadsto enable their recycling and reuse; at least one collection vessel andat least one fourth tangential flow filtration system to allow forconcentration and buffer exchange of the biological product; at leasttwo buffer systems; at least one resin bead recycling buffer system; atleast one sensor or detector; and, at least one fluid handling pump.

The equipment design for the negative charge-based TFF purificationmodule (FIG. 32) enables continuous, biological purification andprovides for a small footprint.

The at least one association equilibration vessel volume and agitationcapabilities of the negative charge-based TFF purification moduleconsider the input flow rate and throughput, equilibration time andagitation rate to enable association kinetics, and resin beadconcentration and charge or electrostatic association capacity. Theassociation buffer for the negative charge-based TFF purification moduleis dependent on the biological product of interest (e.g., a monoclonalantibody) and the small impurities to be removed. Considerations for thebuffer include pH, ionic strength, use of surfactants, or use of organicand/or inorganic salts, specifically to maintain favorable charge orelectrostatic interactions between the target monoclonal antibody andnegatively charged bead surface.

The hollow fiber membrane filter length and surface area of the negativecharge-based TFF purification module is dependent on the solutionviscosity, small impurities and resin bead concentration, and input flowrate and throughput volume. The hollow fiber membrane material isselected from low protein binding materials, including, but not limitedto, PES, mPES, or MCE. The pore size of the hollow fiber membrane isselected from the range of about 10 kDa to about 1 μm. The innerdiameter of the hollow fiber membrane is selected from the range ofabout 0.5 mm to about 5 mm.

The resin bead selection of the negative charge-based TFF purificationmodule is important for negligible leachables and robust stability toenable recycling and reuse in the negative charge-based purificationmodule.

The anionic surface selection for the negative charge-based TFFpurification module is important and may include anionic polymers, netnegatively charged peptides or proteins, carboxyl functionality, andselection is based on achieving appropriate charge or electrostaticinteractions and association stability between the negatively chargedbead surface and the biological product within a defined buffer (pH andionic strength).

The at least one dissociation equilibration vessel volume and agitationcapabilities of the negative charge-based TFF purification moduleconsider the input flow rate and throughput, equilibration time toenable dissociation kinetics, and a dissociation buffer, for example, adissociation buffer at 10× to enable dilution during equilibration timeto arrive at 1× final buffer salt concentration. The dissociation bufferis dependent on the strength of the charge or electrostatic interactionsbetween the biological product of interest (e.g., a monoclonal antibody)and the resin bead anionic surface, and variations may include pH, ionicstrength, use of surfactants, or use of organic and/or inorganic salts.In some aspects, multiple dissociation equilibration vessels areutilized with multiple tangential flow filtration systems to achieve agradient dissociation, for example, a pH gradient or an ionic strengthgradient.

The at least one regeneration equilibration vessel of the negativecharge-based TFF purification module is used in combination with the atleast one third tangential flow filtration system to allow forconcentration and buffer exchange of the resin beads to return the resinbeads to their initial condition, thus, enabling recycling and reuse ofthe resin beads.

The at least one collection vessel of the negative charge-based TFFpurification module is used in combination with the at least one fourthtangential flow filtration system to allow for concentration and bufferexchange of the fraction containing the biological product.

In embodiments, the at least one association equilibration vessel, theat least one dissociation equilibration vessel, and the at least oneregeneration equilibration vessel of the negative charge-based TFFpurification module may comprise a single vessel that is transitionedbetween the corresponding tangential flow filtration systems to enablepurification and regeneration of the resin beads with appropriatebuffers, while maintaining continuous flow of the filtrate via at leastone additional vessel on a parallel flow path.

In embodiments, the regeneration of the resin beads may be accomplishedwith solely the at least one dissociation equilibration vessel and theat least one second tangential flow filtration system of the negativecharge-based TFF purification module configured to comprise both the lowpH elution buffer and the regeneration buffer to enable purification,concentration and buffer exchange, thus regenerating the resin beadswithout necessitating a separate regeneration equilibration vessel andcorresponding tangential flow filtration system.

Viral inactivation and/or removal may be accomplished during theassociation or dissociation buffer equilibration and subsequent fluidicprocessing steps depending on buffer composition and pH.

The negative charge-based TFF purification module may include in-linesampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques. Further, the in-line analyticalmeasurement techniques (e.g., flow sensors, pressure sensors, opticaldensity measurement devices, UV detectors, RI detectors) may be used toenable feedback control mechanisms with the process.

Unlike the traditional packed column chromatography and column-switchingchromatography methods commonly used in the art, the negativecharge-based TFF purification module utilizes a mobile affinity resincapable of in situ regeneration and recycling to enable more efficientuse of the resin and to enable continuous processing in asmall-footprint without concerns of traditional column capacitylimitations.

Isoelectric Point-Based, Fluidic Purification Module

As provided herein, an isoelectric point-based, fluidic purificationmodule for separating a mixture into two or more fractions, at least onefraction containing a biological product is described. The isoelectricpoint-based, fluidic purification module includes at least one inlet andat least one outlet configured to permit continuous fluid flow betweenthe at least one inlet and the at least one outlet and wherein the flowrate may be, for example, consistent and constant during steady-stateoperation.

In embodiments, the isoelectric point-based fluidic purification moduleincludes at least one free-flow electrophoresis apparatus comprising afluidic device (e.g., a mesofluidic, a millifluidic, a macrofluidicdevice, or any combination thereof) having a fluidic channel createdbetween two parallel plates; an electric field or electric fieldgradient orthogonal to the fluid flow direction; an aqueous ionicsolution (FIG. 33); at least one de-bubbling or de-gassing system toremove electrolysis bubbles near the point of generation by a vacuumsystem to create a bubble-free main separation channel and to enablecontinuous, long-term operation; at least one liquid circuit breaker; atleast one buffer or ampholyte system; at least one electrode solution;at least one sensor or detector; at least one fluid handling pump; andat least one collection vessel.

In other embodiments, the isoelectric point-based, fluidic purificationmodule includes at least one first free-flow electrophoresis apparatuscomprising a fluidic device (e.g., a mesofluidic, a millifluidic, amacrofluidic device, or any combination thereof) having a fluidicchannel created between two parallel plates, an electric field orelectric field gradient orthogonal to the fluid flow direction, anaqueous ionic solution; and at least one second free-flowelectrophoresis apparatus comprising a fluidic device (e.g., amesofluidic, a millifluidic, a macrofluidic device, or any combinationthereof) having a fluidic channel created between two parallel plates,an electric field or electric field gradient orthogonal to the fluidflow direction, and an aqueous ionic solution; wherein each free-flowelectrophoresis apparatus is connected in series and is capable ofoperating in an independent mode of operation to enable purification(FIGS. 34-36). For example, without intent to be limiting, the at leastone first free-flow electrophoresis apparatus may operate in anisoelectric focusing mode and the at least one second free-flowelectrophoresis apparatus may operate in an isotachophoresis mode toincrease separation resolution.

In embodiments, the isoelectric point-based, fluidic purification modulecomprises at least one first free-flow electrophoresis apparatuscomprising a fluidic device (e.g., a mesofluidic, a millifluidic, amacrofluidic device, or any combination thereof) having a fluidicchannel created between two parallel plates, an electric field orelectric field gradient orthogonal to the fluid flow direction, and acoarse pH gradient across the main separation channel (e.g., a pH rangefrom about 2 to about 10); and at least one second free-flowelectrophoresis apparatus comprising a fluidic device (e.g., amesofluidic, a millifluidic, a macrofluidic device, or any combinationthereof) having a fluidic channel created between two parallel plates,an electric field or electric field gradient orthogonal to the fluidflow direction, and a fine pH gradient across the main separationchannel (e.g., a pH range from about 5 to about 8); at least onede-bubbling or de-gassing system; at least one liquid circuit breaker;at least one buffer or ampholyte system; at least one electrodesolution; at least one collection vessel; at least one sensor ordetector; and at least one fluid handling pump (FIG. 34). In examples,the at least one first free-flow electrophoresis apparatus and the atleast one second free-flow electrophoresis apparatus are connected inseries and operated in an isoelectric focusing modes.

In embodiments, the isoelectric point-based, fluidic purification modulecomprises at least one first free-flow electrophoresis apparatuscomprising a fluidic device (e.g., a mesofluidic, a millifluidic, amacrofluidic device, or any combination thereof) having a fluidicchannel created between two parallel plates, an electric field orelectric field gradient orthogonal to the fluid flow direction, andconstant basic pH across the main separation channel with no pH gradient(e.g., a pH of greater than 7); and at least one second free-flowelectrophoresis apparatus comprising a fluidic device (e.g., amesofluidic, a millifluidic, a macrofluidic device, or any combinationthereof) having a fluidic channel created between two parallel plates,an electric field or electric field gradient orthogonal to the fluidflow direction, and a constant acidic pH across the main separationchannel with no pH gradient (e.g., a pH of less than 7); at least onede-bubbling or de-gassing system; at least one liquid circuit breaker;at least one buffer or ampholyte system; at least one electrodesolution; at least one collection vessel; at least one sensor ordetector; and at least one fluid handling pump (FIG. 35). In examples,the at least one first free-flow electrophoresis apparatus and the atleast one second free-flow electrophoresis apparatus are connected inseries and operated in zone electrophoresis modes.

In embodiments, the isoelectric point-based, fluidic purification modulecomprises at least one first free-flow electrophoresis apparatuscomprising a fluidic device (e.g., a mesofluidic, a millifluidic, amacrofluidic device, or any combination thereof) having a fluidicchannel created between two parallel plates, an electric field orelectric field gradient orthogonal to the fluid flow direction, and a pHgradient across the main separation channel (e.g., a pH range from about4 to about 9); and at least one second free-flow electrophoresisapparatus comprising a fluidic device (e.g., a mesofluidic, amillifluidic, a macrofluidic device, or any combination thereof)comprising a fluidic channel created between two parallel plates, anelectric field or electric field gradient orthogonal to the fluid flowdirection, and both an acidic pH gradient and a basic pH gradientseparated by a spacer solution (e.g. NaCl solution); at least onede-bubbling or de-gassing system; at least one liquid circuit breaker;at least one buffer or ampholyte system; at least one electrodesolution; at least one collection vessel; at least one sensor ordetector; and at least one fluid handling pump (FIG. 36). In examples,the at least one first free-flow electrophoresis apparatus and the atleast one second free-flow electrophoresis apparatus are connected inseries and operated in an isoelectric focusing mode and anisotachophoresis mode, respectively.

Alternatively, the isoelectric point-based, fluidic purification moduleincludes at least one first fluidic device (e.g., a mesofluidic, amillifluidic, a macrofluidic device, or any combination thereof)comprising a fluidic channel having at least one dielectrophoreticelectrode capable of inducing a defined, unidirectional force; at leastone second fluidic device comprising a free-flow electrophoresisapparatus having a fluidic channel created between two parallel plates,an electric field or electric field gradient orthogonal to the fluidflow direction, and a pH gradient across the main separation channel(e.g., a pH range from about 4 to about 9); and at least one thirdfluidic device comprising a free-flow electrophoresis apparatus having afluidic channel created between two parallel plates, an electric fieldor electric field gradient orthogonal to the fluid flow direction, andboth an acidic pH gradient and a basic pH gradient separated by a spacersolution (e.g. NaCl solution); at least one de-bubbling or de-gassingsystem; at least one liquid circuit breaker; at least one buffer orampholyte system; at least one electrode solution; at least onecollection vessel; at least one sensor or detector; and at least onefluid handling pump (FIG. 37). In examples, the at least one firstfluidic device having at least one selective dielectrophoreticelectrode, the at least one second fluidic device comprising a free-flowelectrophoresis apparatus and the at least one third fluidic devicecomprising a free-flow electrophoresis apparatus are connected in seriesand operated in manner to pre-sort the mixture containing a biologicalproduct prior to purification via an isoelectric focusing mode and anisotachophoresis mode by the second and third fluidic devices or chips,respectively.

In embodiments, additional, subsequent free-flow electrophoresisapparatuses comprising a fluidic device (e.g., a mesofluidic, amillifluidic, a macrofluidic device, or any combination thereof) havinga fluidic channel created between two parallel plates, an electric fieldor electric field gradient orthogonal to the fluid flow direction, andan aqueous ionic solution may be used to enable enhanced separationresolution. For example, without intent to be limiting, an additionalfree-flow electrophoresis apparatus having an ampholyte solution capableof generating a refined pH gradient across the main separation channel(e.g., a pH range from about 7.1 to about 7.6), may be used to increasethe separation resolution of a monoclonal antibody.

The equipment design for the isoelectric point-based, fluidicpurification module (FIGS. 33-37) enables continuous, biologicalpurification and provides for a small footprint.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least two electrodes (e.g. platinum wireelectrodes) to function as an anode or a cathode.

In embodiments, the isoelectric point-based, fluidic purificationapparatus includes at least one electrode solution. In some embodiments,the at least one electrode solution comprises an electrolyte solutionconfigured to contact and enable the appropriate function of an anode ora cathode, for example, sulfuric acid and sodium hydroxide,respectively. In other embodiments, the at least one electrode solutioncomprises the same ampholyte composition as is present in the mainseparation channel configured to enable the appropriate function of ananode or a cathode, for example, Tris buffered saline flowing throughthe main separation channel, the anode channel, and the cathode channel.

In embodiments, the isoelectric point-based fluidic purificationapparatus further comprises at least one de-bubbler system tocontinuously remove, via a vacuum system, O₂ and H₂ gas bubbles thatevolve in the electrode channels under applied voltage (FIG. 38). Insome embodiments, removal of electrolysis bubbles is essential to enablecontinuous operation for substantially long periods of time. Removal ofthe electrolysis bubbles directly from the electrode channels creates abubble-free main separation channel. In examples, the de-bubbler systemutilizes a hydrophobic PTFE membrane to create a water-tight seal atopthe electrode channel that permits continuous removal of electrolysisbubbles at the point of generation by exposure to a vacuum system. Inexamples, the vacuum gauge pressure ranges from about −0.05 bar to about−0.4 bar.

The fluidic channel dimensions and the applied voltage across thechannel to generate the orthogonal electric field or electric fieldgradient to enable protein separation at high flow rates (e.g., greaterthan 1 mL/min) may necessitate the implementation of a robust activecooling system or heat sink to dissipate Joule heat and maintain desiredoperating temperatures, for example, between about 4° C. and about 50°C., preferably from 4° C. to about 37° C., to ensure thermostability ofthe biological product (e.g., a monoclonal antibody). For example, theactive cooling system may comprise an aluminum thermal chuck containinga chilled, circulating water/propylene glycol jacket with a feedbackcontrol loop to maintain a constant temperature ranging from about 10°C. to about 25° C. Further, the dimensions of the channel are criticalparameters to enable adequate protein separation and is dependent on thenumber of proteins to be separated and their range of their respectiveisoelectric points.

The backpressure within the isoelectric point-based fluidic purificationapparatus is dependent on the channel geometry and dimensions, the inletand outlet opening and/or tubing diameters, and the input flow rate. Inexamples, the backpressure ranges from about 0.5 psi to about 10 psi. Insome examples, the backpressure is controlled by, for example, withoutintent to be limiting, a needle valve.

In order to perform in-line sensing and detection, for example, with aflow sensor, a temperature sensor, a conductivity sensor, a pH sensor, arefractive index detector, a UV detector, a backpressure sensor, or anycombination thereof, the solution must be voltage-free. In embodiments,the isoelectric point-based, fluidic purification module includes atleast one liquid circuit breaker or disconnect downstream of the fluidicdevice and upstream of the at least one in-line sensor or detector toensure the ability to perform sensing or detection in a voltage-freesolution (FIG. 39).

The throughput of the isoelectric point-based, fluidic purificationmodule may be increased by multiplexing multiple free-flowelectrophoresis apparatuses, in series or in parallel. Moreover,multiple fluidic devices or chips in series or in parallel may berequired to enable adequate purification. Viral inactivation and removalmay be accomplished during the isoelectric point-based fluidicprocessing steps.

The isoelectric point-based, fluidic purification module may includein-line sampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques are contemplated. Further, the in-lineanalytical measurement techniques (e.g., flow sensors, temperaturesensors, pH sensors, conductivity sensors, pressure sensors, opticaldensity measurement devices, UV detectors, RI detectors) may be used toenable feedback control mechanisms with the process.

Continuous Dynamic Filtration Approaches

The transport velocity of the rolled filter membrane across the membranesupport structure may be constant or may change in response to afeedback mechanism (e.g., rotary encoder, a traction encoder wheel) thataccounts for differences between the feed reel and the collection reeldiameter arising from changes in the filter membrane thickness ordiameter during operation. Alternatively, the transport of the rolledfilter membrane across the membrane support structure may be stepped,wherein the vacuum is removed during the stepping and then reappliedfollowing the stepping. In this mode of operation, the at least oneoutput head may have an xy rastering or rθ rastering capability, motionalong the z-axis, and/or at least one additional dynamic filtrationapparatus operating in parallel may be utilized to maintain continuousoperation. Moreover, the stepping phenomenon may also be accomplished byhaving a pick and place robotics system place individual membranes(e.g., a circular membrane piece) onto the membrane support structure,wherein the vacuum is removed during the pick and place mechanics andthen reapplied following the placement. In this mode of operation, theat least one output head may have an xy rastering or rθ rasteringcapability, motion along the z-axis, and/or at least one additionaldynamic filtration apparatus operating in parallel may be utilized tomaintain continuous operation. Furthermore, modes of operation in whichthe filtrate is generated through utilizing positive pressure to drive aheterogeneous mixture across the filter membrane are also contemplatedherein. For example, without intent to be limiting, when the dynamicfiltration apparatus comprises a pick and place robotics system to placeindividual membranes, an xy rastering output head with motion along thez-axis may be used to make contact with the membrane surface to force aheterogeneous mixture across the membrane. Methods of pre-wetting thefilter membrane to increase transport across the membrane is alsocontemplated herein. The at least one vacuum collection vessel may beused until full and subsequently equilibrated to atmospheric pressure ormay be continuously emptied while under vacuum during operation.Additionally, the use of at least one additional dynamic filtrationapparatus operated in parallel to enable continuous processing is alsocontemplated herein. Similarly, the dynamic filtration apparatus maycomprise multiple feed reels of different types of filter membrane(e.g., material and/or pore size) that can be layered and transported atthe same velocity across the active target region to provide enhancedfiltration.

Continuous Loop Conveyor Apparatus Approaches

The at least one magnetic field in the continuous loop conveyorapparatuses performing the affinity-based, magnetic purification and/orcharge-based, magnetic purification steps may be generated by anelectromagnet or a permanent magnetic (e.g., a Neodymium magnet). Themagnetic field may be applied as an on/off toggle or as permanently on(FIGS. 14, 15, 16, 17). When the magnetic field is generated by anelectromagnet, on/off toggling may be accomplished by turning theelectromagnet on or off. When the magnetic field is generated by apermanent magnet, on/off toggling may be accomplished by mechanicallychanging the placement of the magnet from far to within close proximity,for example, within 5 mm of the transport vessel wall surface.Alternatively, when the magnetic field is generated by a permanentmagnet, on/off toggling may be accomplished by transport the vessel intoand out of close proximity (e.g., within 5 mm) of the magnet. Further,the loop conveyor apparatuses may include multiple, discrete magneticfield locations, wherein said discrete magnetic field locations withinthe apparatuses may further require shielding. Additionally, mixing ofthe magnetic resin beads may be accomplished by placing the at least onetransport vessel between two separate and opposing magnetic fields thattoggle between states of on and off. Additionally, the use of at leastone additional affinity-based, magnetic purification or charge-based,magnetic purification apparatus connected in parallel to enablecontinuous processing is also contemplated herein.

Continuous Pick and Place Robotics Apparatus Approaches

The at least one magnetic field in the continuous pick and placerobotics apparatuses performing the affinity-based, magneticpurification and/or charge-based, magnetic purification steps may begenerated by an electromagnet or a permanent magnetic (e.g., a Neodymiummagnet). The magnetic field may be applied as an on/off toggle or aspermanently on (FIGS. 18 and 19). When the magnetic field is generatedby an electromagnet, on/off toggling may be accomplished by turning theelectromagnet on or off. When the magnetic field is generated by apermanent magnet, on/off toggling may be accomplished by mechanicallychanging the placement of the magnet from far to within close proximity,for example, within 5 mm of the transport vessel wall surface.Alternatively, when the magnetic field is generated by a permanentmagnet, on/off toggling may be accomplished by transport the vessel intoand out of close proximity (e.g., within 5 mm) of the magnet. Further,the loop conveyor apparatuses may include multiple, discrete magneticfield locations, wherein said discrete magnetic field locations withinthe apparatuses may further require shielding. Additionally, mixing ofthe magnetic resin beads may be accomplished by placing the at least onetransport vessel between two separate and opposing magnetic fields thattoggle between states of on and off. Additionally, the use of at leastone additional affinity-based, magnetic purification or charge-based,magnetic purification apparatus connected in parallel to enablecontinuous processing is also contemplated herein.

Continuous Mechanical Rotary System Apparatus Approaches

A seal between the at least one gasketed lid and vessel assembly of thecontinuous mechanical rotary apparatus performing the affinity-basedpurification and/or charge-based purification steps system may be formedby a mechanism designed to compress the gasket and ensure the lid isfixed in a sealed 3-dimensional geometry (FIGS. 21-23). In embodiments,the mechanism used to form the seal is a clamping system or interlockingsystem. In some embodiments, the mechanism used to form the seal is ascrew cap system. In other embodiments, the seal is formed bymechanically pressing the lid down uniformly onto the vessel from thetop with a motorized system. In aspects, a mechanical system or arobotics system may be utilized to ensure that the seal is reproduciblyformed and broken in a reversible and repetitive manner over multiplecycles to enable continuous operation. For example, a leak test afterpressurization can be used to analyze the integrity of the seal.Further, the buffer inlets of the at least one lid may be positioned tocreate a circular or vortex-like mixing flow pattern. Additionally, theuse of at least one additional affinity-based purification orcharge-based purification apparatus connected in parallel to enablecontinuous processing is also contemplated herein.

Continuous Staged Linear System Apparatus Approaches

The vessels in a staged linear system are configured to receivecontinuous input flow. The process steps of binding, washing, elution,and regeneration (for affinity-based purification) and association,washing, dissociation, and regeneration (for charge-based purification)are accomplished via connection of each vessel to manifold capable ofautomated liquid handling (FIGS. 24-25). In embodiments, each vessel inthe staged linear system may be configured to perform different processsteps at the same time to enable continuous operation. For example,without intent to be limiting, one vessel may be performing a bindingprocess step while another vessel is performing an elution process step.

Continuous Hybrid Fluidic Device Approaches

The at least one magnetic field in the continuous hybrid fluidic device(FIG. 28) performing the affinity-based, fluidic purification and/orcharge-based, fluidic purification steps may be generated by anelectromagnet, a permanent magnet (e.g., a Neodymium magnet) or apatterned magnet. The independent permanent magnets may require localshielding. Moreover, to achieve high flow rates, the combination of themagnetic components and the piezoelectric components or the combinationof the magnetic components and the dielectrophoretic components mayrequire precise placement. Additional cross-flow channel designs arecontemplated. Alternatively, T-junction channel designs are alsocontemplated. Moreover, a coarse/fine screen approach to select abiological product of interest (e.g., a monoclonal antibody) based onisoelectric point. Further, multiple elutions or dissociations isconsidered. The equilibration vessels may be batch, fed-batch,continuous feed and bleed vessels, or plug flow reactors with theappropriate residence times. Additionally, increasing the throughput ofthe affinity-based, fluidic purification or the charge-based, fluidicpurification module by multiplexing multiple fluidic devices or chips,in series or in parallel. Moreover, the use of at least one additionalaffinity-based, fluidic purification or charge-based, fluidicpurification apparatus connected in parallel to enable continuousprocessing is also contemplated herein.

Continuous Tangential Flow Filtration Approaches

The equilibration vessels in the affinity-based TFF purification andcharge-based TFF purification modules (FIGS. 31-32) may be batch,fed-batch, continuous feed and bleed vessels, or plug flow reactors withthe appropriate residence times. The use of additional tangential flowfiltration systems to enable continuous operation, buffer exchange,diafiltration, ultrafiltration/diafiltration,microfiltration/diafiltration, and/or concentration is also contemplatedherein.

Continuous Free-flow Electrophoresis Approaches

An electric field orthogonal to the direction of fluid flow may be usedfor purification of a biological product (e.g., a monoclonal antibody)in an aqueous ionic solution and/or pH gradient, for example, in thefree-flow electrophoresis apparatus comprising a fluidic channel createdbetween two parallel plates, an electric field or electric fieldgradient orthogonal to the fluid flow direction, and an aqueous ionicsolution and/or pH gradient, as described herein (FIGS. 33-37). Thefluidic channel dimensions are dependent on flow rate, throughputvolume, time to achieve separation, diffusive band broadening, magnitudeof the electric field, and are further dependent on cooling systemcapabilities to dissipate Joule heat. Moreover, the channel design mayhave multiple inlets to generate a pH gradient by delivering one or morebuffer or ampholyte systems. An applied voltage may be dependent on theflow rate, pH gradient, the biological product charge and/or isoelectricpoint (e.g., monoclonal antibody isoelectric point), or the coolingsystem capabilities to dissipate Joule heat. Various buffer and/orampholyte systems are dependent on the biological product of interest(e.g., monoclonal antibody of interest) and its unique isoelectric point(pI) and are thus selected to modulate its charge. Control of pH andionic strength and use of organic salts, inorganic salts, acids, bases,zwitter-ions, and/or ampholytes within the buffer and/or ampholytesystem to establish a continuous pH gradient effect across the channelis contemplated. Alternatively, the pH gradient may be stratified viamultiple inlets delivering discrete buffer and/or ampholyte systems toachieve a desired gradient, for example, without intent to be limiting,a continuous gradient or a stepwise gradient. Moreover, a combination ofcoarse and fine pH gradients may be necessary to purify solely thebiological product (e.g., monoclonal antibody). Considerations regardinga robust cooling mechanism to enable sufficient heat transfer (e.g., aPeltier device, a thermal chuck with a circulating water/propyleneglycol jacket) are also contemplated. Multiplexing of free-flowelectrophoresis apparatuses may accommodate higher input flow rates, andthus higher throughput. Also, the potential to further remove residualinactivated virus during the isoelectric point-based, fluidicpurification steps is contemplated.

Incorporation of Standard Semi-Continuous, Industry Downstream Processes

To enable a turn-key, end-to-end process (e.g., from bioreactor-basedmonoclonal antibody production through obtaining the biological productin final form), standard semi-continuous industry downstream processequipment (and steps) may be further added to the process describedherein to further purify, buffer exchange, and concentrate the purifiedbiological product (e.g., a monoclonal antibody) (FIGS. 48 and 49).

A designated virus inactivation and filtration step may or may not benecessary if virus inactivation and removal is adequately accomplishedby modules (e.g., wherein LRV >4 for MVM and MuLV viruses). That said,the addition of a designated virus inactivation and filtration step iscontemplated herein.

Depending on the purity of the biological product (e.g., monoclonalantibody purity) achieved by the continuous process modules describedherein, off-the-shelf tangential flow filtration (TFF),ultrafiltration/diafiltration (UF/DF), or high performance tangentialflow filtration (HP-TFF) technologies run in fed-batch or perfusion modemay be used to further purify, buffer exchange, or concentrate the finalbiological product prior to fill-finish operations, which may include,vial filling, lyophilization, filter sterilization, terminalsterilization, or combinations thereof. Moreover, in-line sampling portsfor in-process analytical testing and/or in-line analytical measurementtechniques are contemplated.

Methods

Provided herein are methods of continuously purifying a biologicalproduct from a heterogeneous mixture derived from a bioreactor producingsaid biological product at steady-state comprising utilizing the processdescribed herein. As used herein, the terms “steady-state” or “dynamicequilibrium” may refer to a system or process that remains steady overtime in the presence or absence of perturbations. For example, abioreactor that produces said biological product at steady-stateprovides that the expression host cell, for example, a mammalian cell(e.g., CHO cell) or a bacterial cell (e.g., E. coli) can be grown in aphysiological steady-state under constant environmental conditions. Inthis steady state, growth occurs at a constant specific cell culturegrowth rate and all culture parameters remain constant (culture volume,dissolved oxygen concentration, nutrient and product concentrations, pH,cell density, etc.). In addition, environmental conditions can becontrolled by the feedback mechanisms (e.g., a feed/bleed system)inherent to the bioreactor (e.g., a fed-batch, a perfusion, or achemostat bioreactor) in order to maintain the steady-state productionof the biological product over time. The cell density, for example,remains constant over time. In other examples, the protein concentrationremains constant overtime.

A method of continuously purifying a biological product from aheterogeneous mixture derived from a bioreactor producing saidbiological product at steady-state comprising utilizing at least one ofthe modules described herein (for example, the dynamic filtrationmodule, the affinity-based, magnetic purification module, the positivecharge-based, magnetic purification module, the negative charge-based,magnetic purification module, the affinity-based purification module,the positive charge-based purification module, the negative charge-basedpurification module the affinity-based, fluidic purification module, thepositive charge-based, fluidic purification module, the negativecharge-based, fluidic purification module, the affinity-based TFFpurification module, the positive charge-based TFF purification module,the negative charge-based TFF purification module, and/or theisoelectric point-based, fluidic purification module) is disclosed.

Additionally, a method of purifying a biological product from aheterogeneous mixture derived from a bioreactor producing saidbiological product comprising utilizing at least one of the modulesdescribed herein (for example, the dynamic filtration module, theaffinity-based, magnetic purification module, the positive charge-based,magnetic purification module, the negative charge-based, magneticpurification module, the affinity-based purification module, thepositive charge-based purification module, the negative charge-basedpurification module, the affinity-based, fluidic purification module,the positive charge-based, fluidic purification module, the negativecharge-based, fluidic purification module, the affinity-based TFFpurification module, the positive charge-based TFF purification module,the negative charge-based TFF purification module, and/or theisoelectric point-based, fluidic purification module) is also disclosed.For example, at least one of the modules described herein (for example,the dynamic filtration module, the affinity-based, magnetic purificationmodule, the positive charge-based, magnetic purification module, thenegative charge-based, magnetic purification module, the affinity-basedpurification module, the positive charge-based purification module, thenegative charge-based purification module, the affinity-based, fluidicpurification module, the positive charge-based, fluidic purificationmodule, the negative charge-based, fluidic purification module, theaffinity-based TFF purification module, the positive charge-based TFFpurification module, the negative charge-based TFF purification module,and/or the isoelectric point-based, fluidic purification module) mayreplace or be used in addition to a traditional purification techniqueutilized in current batch, single-use, or semi-continuous processesknown in the art.

Alternatively, a method of purifying a biological product from a mixturenot derived from a bioreactor producing said biological productcomprising utilizing at least one of the modules described herein (forexample, the dynamic filtration module, the affinity-based, magneticpurification module, the positive charge-based, magnetic purificationmodule, the negative charge-based, magnetic purification module, theaffinity-based purification module, the positive charge-basedpurification module, the negative charge-based purification module, theaffinity-based, fluidic purification module, the positive charge-based,fluidic purification module, the negative charge-based, fluidicpurification module, the affinity-based TFF purification module, thepositive charge-based TFF purification module, the negative charge-basedTFF purification module, and/or the isoelectric point-based, fluidicpurification module) is also disclosed. For example, at least one of themodules described herein (for example, the dynamic filtration module,the affinity-based, magnetic purification module, the positivecharge-based, magnetic purification module, the negative charge-based,magnetic purification module, the affinity-based purification module,the positive charge-based purification module, the negative charge-basedpurification module, the affinity-based, fluidic purification module,the positive charge-based, fluidic purification module, the negativecharge-based, fluidic purification module, the affinity-based TFFpurification module, the positive charge-based TFF purification module,the negative charge-based TFF purification module, and/or theisoelectric point-based, fluidic purification module) may replace or beused in addition to a traditional purification technique utilized incurrent batch, single-use, or semi-continuous processes known in theart.

As described herein, the term “module” or “modular” may refer toseparate distinct parts (e.g., the dynamic filtration module, theaffinity-based, magnetic purification module, the positive charge-based,magnetic purification module, the negative charge-based, magneticpurification module, the affinity-based, fluidic purification module,the positive charge-based, fluidic purification module, the negativecharge-based, fluidic purification module and/or the isoelectricpoint-based, fluidic purification module) that may be used alone, or inany combination. Moreover, the process may include one or more of any ofthe above-described modules. The term “modular” may refer to and mean astructure which is constructed from a plurality of modular units andwhich may be constructed in a wide variety of structural forms. Forexample, the modular units can be connected together in the form of atransport system or a continuous fluid handling system. Moreover,in-line sampling ports for in-process analytical testing and/or in-lineanalytical measurement techniques are contemplated within each of themodules.

General Definitions

The following definitions are included for the purpose of understandingthe present subject matter and for constructing the appended patentclaims. The abbreviations used herein have their conventional meaningswithin the chemical and biological arts.

While various embodiments and aspects of the present invention are shownand described herein, it will be obvious to those skilled in the artthat such embodiments and aspects are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, without limitation, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, N Y 1989). Any methods,devices, and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a,” “an,” and “the” areunderstood to be singular or plural.

The term “about” when used in reference to numerical ranges, cutoffs, orspecific values is used to indicate that the recited values may vary byup to as much as 25% from the listed value. As many of the numericalvalues used herein are experimentally determined, it should beunderstood by those skilled in the art that such determinations can, andoften times will, vary among different experiments. The values usedherein should not be considered unduly limiting by virtue of thisinherent variation. The term “about” is used to encompass variations of±25% or less, variations of ±20% or less, variations of 10% or less,variations of ±5% or less, variations of ±1% or less, variations of±0.5% or less, or variations of ±0.1% or less from the specified value.About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwiseclear from the context, all numerical values provided herein aremodified by the term “about.”

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 60 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50,as well as all intervening decimal values between the aforementionedintegers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,and 1.9. With respect to sub-ranges, “nested sub-ranges” that extendfrom either end point of the range are specifically contemplated. Forexample, a nested sub-range of an exemplary range of 1 to 60 maycomprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

The term “subject” as used herein refers to a living member of theanimal kingdom. In embodiments, the subject is a member of a speciescomprising individuals who may naturally suffer from the disease. Inembodiments, the subject is a mammal. Non-limiting examples of mammalsinclude rodents (e.g., mice and rats), primates (e.g., lemurs,bushbabies, monkeys, apes, and humans), rabbits, dogs, horses, cats,livestock (such as pigs, bovines, donkeys, mules, bison, goats, camels,and sheep). In embodiments, the subject is a human.

The terms “within close proximity to” or “within close proximity of” asused herein refers to a distance of less than about 1 cm, for example,less than about 5 mm (meaning, for example, the distance between amagnet or a magnetic field and a transport vessel wall or a cross-flowchannel).

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention.

In the descriptions herein and in the claims, phrases such as “at leastone of” or “one or more of” may occur followed by a conjunctive list ofelements or features. The term “and/or” may also occur in a list of twoor more elements or features. Unless otherwise implicitly or explicitlycontradicted by the context in which it is used, such a phrase isintended to mean any of the listed elements or features individually orany of the recited elements or features in combination with any of theother recited elements or features. For example, the phrases “at leastone of A and B;” “one or more of A and B;” and “A and/or B” are eachintended to mean “A alone, B alone, or A and B together.” A similarinterpretation is also intended for lists including three or more items.For example, the phrases “at least one of A, B, and C;” “one or more ofA, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, Balone, C alone, A and B together, A and C together, B and C together, orA and B and C together.” In addition, use of the term “based on,” aboveand in the claims is intended to mean, “based at least in part on,” suchthat an unrecited feature or element is also permissible.

The terms “mechanically smooth” or “substantially smooth” are usedinterchangeably herein to describe a contact surface of a materialhaving a low static coefficient of friction of about 0.01 to about 0.1.

The terms “hybrid fluidic device” or “hybrid fluidic chip” are usedinterchangeably herein to describe a fluid flow device or chip thatcombines cross-flow channel dynamics, magnetophoretic dynamics, and,either acoustophoretic or dielectrophoretic dynamics together tomanipulate magnetic resin beads at high flow rate. Further, hybridfluidic devices may also include electrokinetic or capillary flowdynamics. In some aspects herein, a hybrid fluidic device or chipcomprises a cross-flow channel, at least one magnetic field, and atleast one piezoelectric component (e.g., piezoelectric crystal). Inother aspects herein, a hybrid fluidic device or chip comprises across-flow channel, at least one magnetic field, and at least onedielectrophoretic electrode. Hybrid fluidic devices or chips may bemicrofluidic, mesofluidic, millifluidic, macrofluidic, or anycombination thereof. For example, without intent to be limiting, ahybrid fluidic device can be a microfluidic device comprising across-flow channel, at least one magnetic field, and at least onedielectrophoretic electrode.

The terms “tangential flow filtration (TFF) system,” “high performancetangential flow filtration (HP-TFF) system,” and “cross-flow filtrationsystem (CFF)” are used interchangeably herein, to refer to an equipmentand controls system wherein a sample solution is fed from a feed vesselin a flow path parallel to a porous membrane face allowing one fractionto pass orthogonally through a membrane (e.g., permeate), while theremainder of the sample solution is recirculated back to the sample feedvessel (e.g., retentate) to enable purification of the sample solutionby microfiltration, ultrafiltration, diafiltration, or any combinationthereof. The membrane may be either a flat plate or hollow fibergeometry, charged or uncharged. Further, tangential flow filtration andcross-flow filtration systems are defined as one-dimensional systemsused to purify biomolecules by separation based on a tenfold differencein hydrodynamic size. In contrast, high performance tangential flowfiltration systems are defined as two-dimensional systems that purifybiomolecules by separation based on both differences in chargecharacteristics and a tenfold difference in hydrodynamic size.

The terms “microfilter,” “microfiltrate” or “microfiltration” as usedherein, refer to a TFF process utilizing a membrane with a pore sizegreater than 0.1 micron to concentrate resin beads.

The terms “ultrafilter” or “ultrafiltrate” or “ultrafiltration” as usedherein, refers to a TFF process utilizing a membrane with a pore sizeless than 0.1 micron to concentrate a biological product (e.g., aprotein or fragment thereof (a polypeptide), an antibody or fragmentthereof, a cytokine, a chemokine, an enzyme, or a growth factor).

The terms “diafilter,” “diafiltrate” or “diafiltration” as used herein,refers to a TFF process in which a retentate produced by microfiltrationor ultrafiltration is diluted with buffer solution and subsequentlyre-microfiltered or re-ultrafiltered, respectively, to further purifythe retentate or enable buffer exchange.

The term “diavolume” as used herein, refers to the volume ofdiafiltration buffer utilized in the unit TFF operation compared to theinitial retentate volume.

The terms “free-flow electrophoresis” and “isoelectric point-based,fluidic purification” are used interchangeably herein to a continuousflow process of separating a biological product based upon its charge,its isoelectric point, its electrophoretic mobility, or any combinationthereof. In some aspects herein, free flow electrophoresis has differentmodes of operation, including, but not limited to, isoelectric focusing(IEF-FFE), zone electrophoresis (ZE-FFE), isotachophoresis, or anycombination thereof.

The term “isoelectric point” or “pI” as used herein refers to the pH atwhich a protein is charge neutral or has no net electrical charge.

A pH gradient, as used herein may refer to a “coarse pH gradient” or a“fine pH gradient.” For example, a coarse pH gradient refers to a pHrange (or gradient) of a pH from about 2 to about 10, or from about 2 toabout 9, or from about 2 to about 8, or from 2 to about 7, or from 2 toabout 5, or from 2 to about 4, or from about 2 to about 3. Moreover, acoarse pH gradient may be a pH range from about 5 to about 8.Alternatively, a fine pH gradient refers to a pH gradient (e.g., a pHchange) within 1 pH unit. For example, the pH range may be from about7.0 to about 8.0, or from about 7.1 to about 8.0, or from about 7.2 toabout 8.0, or from about 7.3 to about 8.0, or from about 7.4 to about8.0, or from about 7.4 to about 8.0, or from about 7.5 to about 8.0, orfrom about 7.6 to about 8.0, or from about 7.7 to about 8.0, or fromabout 7.8 to about 8.0, or from about 7.9 to about 8.0. Alternatively,the pH range in a fine pH gradient may range from about 7.0 to about7.5, or from about 7.1 to about 7.5, or from about 7.3 to about 7.5, orfrom about 7.4 to about 7.5 Moreover, the pH range may be from about 7.1to about 7.6.

The term “ampholyte” as used herein refers to an amphoteric electrolyteor an electrolyte that has both acid and base functionality. In someaspects herein, ampholytes comprise amphoteric organic buffer salts oramino acids that are utilized to establish a pH gradient in anisoelectric point-based, fluidic purification apparatus. For example,without intent to be limiting, ampholytes comprise, Tris, HEPES, MES,glycine, histidine, arginine, glutamic acid, or any combination thereof.

The term “antibody” herein is used in the broadest sense and encompassespolypeptides or proteins that comprise or consist of antibody domains,which are understood as constant and/or variable domains of the heavyand/or light chains of immunoglobulins, with or without a linkersequence. The term encompasses various antibody structures, includingbut not limited to monoclonal antibodies, polyclonal antibodies,multi-specific antibodies such as bispecific antibodies, and antibodyfragments. The term “monoclonal antibody” as used herein refers toidentical antibodies or antibody fragments produced by a single clone ofcells or cell line that have binding affinity and specificity for atarget antigen. Antibody domains may be of native structure or modifiedby mutagenesis or derivatization. Further, the term “immunoglobulin”refers to a protein having the structure of a naturally occurringantibody. For example, immunoglobulins of the IgG class areheterotetrameric glycoproteins of about 150,000 Daltons, composed of twolight chains and two heavy chains that are disulfide-bonded. From N- toC-terminus, each heavy chain has a variable region (VH), also called avariable heavy domain or a heavy chain variable domain, followed bythree constant domains (CH1, CH2, and CH3), also called a heavy chainconstant region. Similarly, from N- to C-terminus, each light chain hasa variable region (VL), also called a variable light domain or a lightchain variable domain, followed by a constant light (CL) domain, alsocalled a light chain constant region. An immunoglobulin of the IgG classessentially consists of two F(ab) domains and an Fc domain, linked viathe immunoglobulin hinge region. The heavy chain of an immunoglobulinmay be assigned to one of five types, namely, IgG, IgM, IgA, IgE, andIgD immunoglobulin isotypes derived from any animal (e.g., any of theanimals conventionally used, for example, sheep, rabbits, goats, ormice) which may be further divided into subtypes, such as IgG1, IgG2, orIgG2a. Preferably, the antibody comprises a monoclonal antibody (e.g., ahuman monoclonal antibody).

The term “valent” as used herein refers the presence of a specifiednumber of binding sites in an antibody molecule. As such, the terms“bivalent,” “trivalent,” and “multivalent” denote the presence of twobinding sites, three binding sites, and multiple binding sites,respectively, in an antibody molecule. The term “monovalent” as usedherein with respect to a binding site of an antibody shall refer to amolecule comprising only one binding site directed against a targetantigen. The term “valency” is thus understood as the number of bindingsites directed against the same target antigen, either specificallybinding to the same or different epitopes of an antigen.

The term “monospecific antibody” as used herein means the ability of asingle antibody to have the capability to selectively bind to a single,discrete target with specificity and high affinity. The specificity andhigh affinity means that the monospecific antibody predominantly bindsto one discrete target antigen of interest, while manifesting negligiblebinding to other molecules in a sample solution. A specific binding siteis typically not cross-reactive with other targets, however, thespecific binding site may specifically bind to one or more epitopes,isoforms, or variants of the target. The specific binding means thatbinding is selective in terms of target identity, for example, highbinding affinity or avidity. Selective binding is usually achieved ifthe binding constant or binding dynamics to a target antigen ispreferably at least 100-fold, and more preferably at least 1000-foldcompared to binding constant or binding dynamics to an antigen ormolecule which is not the target antigen. As used herein, the term “highaffinity” refers to a binding interaction of antibody with the targetantigen having an equilibrium dissociation constant (K_(D)) less than orequal to 10-6 M (micromolar affinity), preferably less than 10-9 M(nanomolar affinity), and more preferably less than 10-12 M (picomolaraffinity). As used herein, “no substantial cross-reactivity” means thatan antibody does not recognize or specifically bind an antigen differentfrom the actual target antigen of the molecule, particularly whencompared to that target antigen. For example, an antibody may bind lessthan about 10% to less than about 5% to an antigen different from theactual target antigen or may bind said antigen different from the actualtarget antigen at an amount consisting of less than about 10%, 9%, 8%7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%, preferably less thanabout 2%, 1%, or 0.5%, and most preferably less than about 0.2% or 0.1%antigen different from the actual target antigen. Monospecificantibodies can be one of four types, specifically, human, humanized,chimeric, and murine, and can be derived from IgG, IgM, IgA, IgE, IgDimmunoglobulin isotypes and subtypes. Further, monospecific antibodiescan have monovalent, bivalent, trivalent, or multivalent binding sitesfor the target antigen of interest. Similarly, the term “bispecificantibody” as used herein means the ability of a single antibody havingthe capability to selectively bind to two different and discrete targetswith specificity and high affinity for each target independently. Inaspects, the specificity and high affinity means that the bispecificantibody predominantly binds to the two different and discrete targetantigens of interest, while manifesting negligible binding to othermolecules in a sample solution. Moreover, the term “trispecificantibody” as used herein means the ability of a single antibody havingthe capability to selectively bind to three different and discretetargets with specificity and high affinity for each targetindependently. In aspects, the specificity and high affinity means thatthe trispecific antibody predominantly binds to the three different anddiscrete target antigens of interest, while manifesting negligiblebinding to other molecules in a sample solution. Further, multispecificantibodies can include or be formed from antibody fragments.

The term “antibody fragment” as used herein refers to a molecule otherthan an intact antibody that comprises a portion of an intact antibodythat binds the antigen to which the intact antibody binds. Non-limitingexamples of antibody fragments include, monovalent IgG, linearantibodies, single-chain antibody molecules, Fv, scFv, scFv-Fc, F(ab),F(ab)2, scF(ab), F(ab)-scFv fusion, F(ab)-(scFv)2 fusion, F(ab)-scFv-Fc,cross-F(ab), nanobodies, minibodies, diabodies, scFv-Fc diabodies,and/or affibodies. In addition, antibody fragments comprise single chainpolypeptides having the characteristics of a VH domain, namely beingable to assemble together with a VL domain, or of a VL domain, namelybeing able to assemble together with a VH domain to a functional antigenbinding site and thereby providing the antigen binding property offull-length antibodies.

The term “chimeric antibody” refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species, usually prepared by recombinant DNAtechniques. Chimeric antibodies may comprise a rabbit or murine variableregion and a human constant region. Other forms of “chimeric antibodies”are those in which the constant region has been modified or changed fromthat of the original antibody to generate the properties according tothe present invention. Such chimeric antibodies are also referred to as“class-switched antibodies”. Chimeric antibodies are the product ofexpressed immunoglobulin genes comprising DNA segments encodingimmunoglobulin variable regions and DNA segments encoding immunoglobulinconstant regions. Methods for producing chimeric antibodies involveconventional recombinant DNA and gene transfection techniques are wellknown in the art (Morrison S L, et al., PNAS, 81:6851-6855, (1984)).

The term “human antibody” as used herein is an antibody which possessesan amino acid sequence which corresponds to that of an antibody producedby a human or a human cell or derived from a non-human source thatutilizes human antibody repertoires or other human antibody-encodingsequences. This definition of a human antibody specifically excludes ahumanized antibody comprising non-human antigen-binding residues. Asalso mentioned for chimeric and humanized antibodies, the term “humanantibody” as used herein also comprises such antibodies which aremodified in the constant region, for example, by “class switching”.

The terms “recombinant antibody” and “recombinant human antibody”, asused herein, are intended to include all human antibodies that areprepared, expressed, created, or isolated by recombinant means, such asantibodies isolated from an expression host cell, for example, withoutintent to be limiting, a mammalian cell (e.g., HEK 293, NSO or CHO cell)or a bacterial cell (e.g., E. coli), or from an animal (e.g., a mouse)that is transgenic for human immunoglobulin genes or antibodiesexpressed using a recombinant expression vector transfected into a hostcell. Such recombinant human antibodies have variable and constantregions in a rearranged form. The recombinant human antibodies accordingto the present invention have been subjected to in vivo somatichypermutation. Thus, the amino acid sequences of the VH and VL regionsof the recombinant antibodies are sequences that, while derived from andrelated to human germ line VH and VL sequences, may not naturally existwithin the human antibody germ line repertoire in vivo. The term“recombinant” as used herein shall mean “being prepared by geneticengineering” or “the result of genetic engineering”, for example,specifically employing heterologous sequences incorporated in arecombinant vector or recombinant host cell.

The term “humanized antibody” refers to a chimeric antibody comprisingamino acid residues from non-human hypervariable regions and amino acidresidues from human framework regions (FRs) which has undergonehumanization. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regions (e.g.,CDRs) correspond to those of a non-human antibody, and all orsubstantially all of the FRs correspond to those of a human antibody. Ahumanized antibody optionally may comprise at least a portion of anantibody constant region derived from a human antibody. Other forms ofhumanized antibodies encompassed by the present invention are those inwhich the constant region has been additionally modified or changed fromthat of the original antibody to generate the new properties, forexample, Fc receptor binding.

The terms “purify,” “purified,” “purifying,” or “purification” as usedherein refer to methods by which impurities are removed from abiological product (e.g., nucleic acid, protein, antibody products andthe like) in a heterogeneous mixture. In aspects, the impurities arecells, cellular debris, aggregates, host cell proteins, undesiredproteins and peptides, undesired antibodies, undesired nucleic acids andoligonucleotides, viruses, salts, buffer components, surfactants,sugars, metallic contaminants, leachables, media components, and/ornaturally-occurring organic molecules with which it is naturallyassociated. In some aspects, the terms “large impurity” or “largeimpurities” as used herein, refer to cells, cell debris, and/oraggregates. In other aspects, the terms “small impurity” or “smallimpurities” as used herein refer to host cell proteins, undesiredproteins and peptides, undesired nucleic acids and oligonucleotides,viruses, salts, buffer components, surfactants, sugars, metalliccontaminants, leachables, media components, and/or naturally-occurringorganic molecules with which it is naturally associated. Purifiedbiological products are at least 60% by weight (dry weight) the productof interest. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight theproduct of interest. For example, a purified antibody is one that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of thedesired antibody by weight. Purity is measured by any appropriatestandard method, for example, by a column chromatography. Purified alsodefines a degree of sterility that is safe for administration to asubject, e.g., lacking infectious, toxic, or immunogenic agents.Similarly, by “substantially pure” means a biological product (e.g.,nucleic acid, protein, antibody products and the like) that has beenseparated from the components that naturally accompany it. Typically,the biological product (e.g., an antibody, a protein, a polypeptide andthe like) is substantially pure when it is at least 60%, 70%, 80%, 90%,95%, or even 99%, by weight, free from impurities (e.g., cells, cellulardebris, aggregates, host cell proteins, undesired proteins and peptides,undesired antibodies, undesired nucleic acids and oligonucleotides,viruses, salts, buffer components, surfactants, sugars, metalliccontaminants, leachables, media components, and/or naturally-occurringorganic molecules with which it is naturally associated).

The terms “isolate,” “isolated,” or “isolation” as used herein refer tomethods by which a desired biological product (e.g., nucleic acid,protein, antibody products and the like) is specifically selected andseparated from undesired products in a heterogeneous mixture. Further,an “isolated antibody,” as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds CD20 and is substantially free of antibodies that specificallybind antigens other than CD20). Moreover, an isolated antibody may besubstantially free of impurities (e.g., cells, cellular debris,aggregates, host cell proteins, undesired proteins and peptides,undesired antibodies, undesired nucleic acids and oligonucleotides,viruses, salts, buffer components, surfactants, sugars, metalliccontaminants, leachables, media components, and/or naturally-occurringorganic molecules with which it is naturally associated).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acids, wherein thepolymer may in embodiments be conjugated to a moiety that does notconsist of amino acids. The terms also apply to amino acid polymers inwhich one or more amino acid residue is an artificial chemical mimeticof a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymers. A “fusion” or “fusion protein” refers to a chimericprotein encoding two or more separate protein sequences that arerecombinantly expressed or chemically synthesized as a single moiety

The term “nucleic acid” as described herein refers to nucleotides (e.g.,deoxyribonucleotides, ribonucleotides, and 2′-modified nucleotides) andpolymers thereof in either single-, double- or multiple-stranded form,or complements thereof. The terms “polynucleotide,” “oligonucleotide,”“oligo” or the like refer, in the usual and customary sense, to a linearsequence of nucleotides. The term “nucleotide” refers, in the usual andcustomary sense, to a single unit of a polynucleotide, i.e., a monomer.Nucleotides can be ribonucleotides, deoxyribonucleotides, or modifiedversions thereof. Examples of polynucleotides contemplated hereininclude single and double stranded DNA, single and double stranded RNA,and hybrid molecules having mixtures of single and double stranded DNAand RNA. Examples of nucleic acid (e.g., polynucleotides) contemplatedherein include any types of RNA (e.g., mRNA, siRNA, miRNA, and guideRNA) and any types of DNA (e.g., genomic DNA, plasmid DNA, andminicircle DNA), and any fragments thereof. Further, as describedherein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acidoligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acidfragment” and “polynucleotide” are used interchangeably and are intendedto include, but are not limited to, a polymeric form of nucleotidescovalently linked together that may have various lengths, eitherdeoxyribonucleotides and/or ribonucleotides, and/or analogs,derivatives, or modifications thereof. Different polynucleotides mayhave different three-dimensional structures, and may perform variousfunctions, known or unknown. Non-limiting examples of polynucleotidesinclude genomic DNA, a genome, mitochondrial DNA, a gene, a genefragment, an exon, an intron, intergenic DNA (including, withoutlimitation, heterochromatic DNA), double stranded DNA (dsDNA), messengerRNA (mRNA), transfer RNA, enhancer RNA (eRNA), micro RNA, interferingRNA (RNAi), small interfering RNA (siRNA), ribosomal RNA, a ribozyme,cDNA, a recombinant polynucleotide, a branched polynucleotide, aplasmid, a vector, an aptamer, isolated DNA of a sequence, isolated RNAof a sequence, a nucleic acid probe, and a primer. Polynucleotidesuseful in the methods of the disclosure may comprise natural nucleicacid sequences and variants thereof, artificial nucleic acid sequences,or a combination of such sequences.

The term “amino acid,” as used herein, encompasses bothnaturally-occurring amino acids and non-naturally-occurring amino acids.For the purposes of this disclosure, the naturally occurring amino acidscomprises the twenty naturally occurring L-amino acids. These 20 aminoacids can be split into those that have neutral charges, positivecharges, and negative charges. For reference, the “neutral” amino acidsare listed along with their respective three-letter and single-lettercode and polarity: Alanine: (Ala, A) nonpolar, neutral; Asparagine:(Asn, N) polar, neutral; Cysteine: (Cys, C) nonpolar, neutral;Glutamine: (Gln, Q) polar, neutral; Glycine: (Gly, G) nonpolar, neutral;Isoleucine: (Ile, I) nonpolar, neutral; Leucine: (Leu, L) nonpolar,neutral; Methionine: (Met, M) nonpolar, neutral; Phenylalanine: (Phe, F)nonpolar, neutral; Proline: (Pro, P) nonpolar, neutral; Serine: (Ser, S)polar, neutral; Threonine: (Thr, T) polar, neutral; Tryptophan: (Trp, W)nonpolar, neutral; Tyrosine: (Tyr, Y) polar, neutral; Valine: (Val, V)nonpolar, neutral; and Histidine: (His, H) polar, positive (10%) neutral(90%). The “positively” charged amino acids are: Arginine: (Arg, R)polar, positive; and Lysine: (Lys, K) polar, positive. The “negatively”charged amino acids are: Aspartic acid: (Asp, D) polar, negative; andGlutamic acid: (Glu, E) polar, negative. Examples of non-naturallyoccurring amino acids include, but are not limited to, D-amino acids(i.e., an amino acid of an opposite chirality to the naturally-occurringform), N-α-methyl amino acids, C-α-methyl amino acids, β-methyl aminoacids and D- or L-β-amino acids. Other non-naturally occurring aminoacids include, for example, β-alanine (β-Ala), norleucine (Nle),norvaline (Nva), homoarginine (Har), 4-aminobutyric acid (γ-Abu),2-aminoisobutyric acid (Aib), 6-aminohexanoic acid (ε-Ahx), ornithine(orn), sarcosine, α-amino isobutyric acid, 3-aminopropionic acid,2,3-diaminopropionic acid (2,3-diaP), D- or L-phenylglycine,D-(trifluoromethyl)-phenylalanine, and D-p-fluorophenylalanine.

The term “continuous” or “semi-continuous” refers to a process by whichthe production and purification of a biological product is performedsubstantially with or without interruption or with minor interruption orwith unintended interruption for prolonged periods of time. For example,the process transferring the bioreactor bleed solution to the dynamicfiltration module is done without interruption, or with minorinterruption. In other examples, the process of removing impurities froma heterogeneous mixture by dynamic filtration and transferring thefiltrate (containing the biological product) to a first module (e.g.,affinity-based purification module) is done without interruption, orwith minor interruption. Moreover, the process of transferring thesolution from the first module to a second module (e.g., charge-basedpurification) is done without interruption, or with minor interruption.In yet other examples, the product stream from dynamic filtrationthrough the first module, through the second module, and/or subsequentsteps is done without interruption, or with minor interruption. In otherwords, a subsequent unit operation can start processing the productstream before a first unit operation has finished processing the productstream.

As used in reference to the dynamic filtration and/or purificationprocesses (affinity-based, magnetic purification, charge-based, magneticpurification, affinity-based purification, charge-based purification,affinity-based, fluidic purification, charge-based fluidic purification,the affinity-based TFF purification module, the charge-based TFFpurification module, and/or isoelectric point-based purificationprocesses) of the present invention, “continuous” means that theprocesses are physically and logistically integrated so as to permitoperation without interruption of the fluid flow derived from asteady-state bioreactor for a prolonged period of time. The processes ofthe present invention are capable of continuous operation, for example,for prolonged periods ranging from 1 day to several months withoutinterrupting the operation or sequence of the processes. The termcontinuous, as used in reference to the processes of the disclosedinvention, is also understood to mean that a process is not performed ina batch-wise manner or in a truly continuous manner. For example, aprocess comprising a hold-up volume may be deemed continuous if theprocess is able to operate without interrupting the fluid flow derivedfrom the bioreactor bleed line. As used in the present invention, theprocesses are operated for a continuous period greater than 2, 3, 4, 5,6, or 7 days, 2, 3, 4, 5, 6, 7 or 8 weeks, or 3, 4, 5, 6 or more months.

The terms “semi-continuous” and “intermittent” mean that one or more ofthe processes or elements of an integrated system operate in adiscontinuous or batch-wise manner, for example, fed-batch modes ofoperation, while other processes or elements of the integrated systemoperate in a continuous manner.

The methods and processes described herein may be continuous,semi-continuous, or not continuous. Minor (and/or unintendedinterruptions, and/or intended interruptions) interruptions, forexample, may include tears or breaks (e.g., within the filter membraneof the dynamic filtration module). For example, a tear or break in thefilter membrane may include any alteration which affects the integrityof the filter membrane to perform its function. Any blockage orobstruction within any of the ports, tubing, devices, or apparatusesthroughout the system may be considered a minor interruption. Otherminor interruptions contemplated include overfilled containers/vesselsor underfilled vessels/containers. A malfunction of the magnet ormagnetic field used during purification may also constitute a minorinterruption. A malfunction of loop conveyer system used duringpurification may also constitute a minor interruption. A malfunction ofpick and place robotics system used during purification may alsoconstitute a minor interruption. A malfunction of mechanical rotarysystem used during purification may also constitute a minorinterruption. A malfunction of in-line analytical measurementinstruments, for example, sensors or detectors used during purificationmay also constitute a minor interruption. A malfunction of feedbackcontrol mechanisms, for example, PID or closed loop controllers, usedduring purification may also constitute a minor interruption.

The term “integrated,” as used in reference to multiple apparatuses,modules, systems and/or processes, means that the apparatuses, modules,systems and/or processes are physically and logistically connected so asto constitute a unified system capable of operating continuously. In thecontext of the system of the present invention, which is directed to anintegrated continuous or semi-continuous system for producing a purifiedbiological product, an integrated system will connect differentcomponents directly and in a manner sufficient to maintain continuousflow between the different components of the system.

The term “weight percent” or “% (w/w)” refers to a percentage of acomponent in a solution that is calculated on the basis of weight forthe component and the solvent. For example, a 1% (w/w) solution of acomponent would have 1 g of the component dissolved in a 100 g ofsolvent. The term “volume percent” or “% (v/v)” refers to a percentageof a component in a solution that is calculated on the basis of volumefor the component and the solvent. For example, a 1% (v/v) solution of acomponent would have 1 ml of the component dissolved in a 100 ml ofsolvent.

EXAMPLES

The following examples illustrate certain specific embodiments of theinvention and are not meant to limit the scope of the invention.

Embodiments herein are further illustrated by the following examples anddetailed protocols. However, the examples are merely intended toillustrate embodiments and are not to be construed to limit the scopeherein. The contents of all references and published patents and patentapplications cited throughout this application are hereby incorporatedby reference.

Example 1: Method of Continuous Production and Purification ofMonoclonal Antibodies Having a Dynamic Filtration Module, anAffinity-Based, Magnetic Purification Module, and a Free-FlowElectrophoresis Module

Etanercept is continuously produced in a chemostat bioreactor operatingat steady-state at a titre of 4 g/L. The heterogeneous mixturecontaining etanercept, large impurities, and small impurities istransferred to a dynamic filtration module via a single output head incommunication with the input line (perfusion bioreactor bleed line) at aflow rate of 10 mL/min. Large impurities are removed by dynamicfiltration (0.45 μm PES, rolled filter membrane; mechanically smoothmembrane support structure with a parallel slotted opening andtemperature control; a wash zone; a membrane transport velocity of 1mm/sec; a vacuum gauge pressure of −0.9 bar; 2 vacuum collection vesselwith a controllable T-valve) to yield a filtrate containing etanerceptand small impurities. Once the first vacuum collection vessel reachescapacity, the flow is diverted to the second vacuum collection vesseland the first vacuum collection vessel is equilibrate to atmosphericpressure.

The filtrate is transferred from the first vacuum collection vessel(atmospheric pressure equilibrated) to the inlet of the affinity-based,magnetic purification module at a flow rate of 10 mL/min via a tubingconnection and a peristaltic pump. The filtrate enters a thin-walled,transport vessel charged with 2% by weight Protein A-coated magneticresin beads (40 μm), suspended in a binding/wash buffer (0.025 M Tris,0.15 M NaCl; pH 7), at the home position of a loop conveyor system. Oncethe transport vessel is filled, the transport vessel moves to anequilibration zone to bind for 30 minutes, while the next transportvessel continues to receive the continuous filtrate flow. Following thebinding of antibodies to Protein A-coated magnetic resin beads, thetransport vessel moves to a permanent magnetic field zone to allow themagnetic resin beads to migrate toward the wall of the transport vessel.The solution containing small impurities is removed by aspiration andsent to a waste vessel. Binding/wash buffer (0.025 M Tris, 0.15 M NaCl;pH 7) is added and the transport vessel moves to an equilibration zoneto enable washing. The transport vessel moves to a permanent magneticfield zone to allow the magnetic resin beads to mix between two separateand opposing magnetic fields that toggle between states of on and offand subsequently migrate toward a single wall of the transport vessel.The solution is removed by aspiration and sent to a waste vessel.Binding/wash buffer is added and the transport vessel moves to anequilibration zone to enable washing. The transport vessel moves to apermanent magnetic field zone to allow the magnetic resin beads to mixbetween two separate and opposing magnetic fields that toggle betweenstates of on and off and subsequently migrate toward a single wall ofthe transport vessel. The solution is removed by aspiration and sent toa waste vessel. Binding/wash buffer is added and the transport vesselmoves to an equilibration zone to enable washing. The transport vesselmoves to a permanent magnetic field zone to allow the magnetic resinbeads to mix between two separate and opposing magnetic fields thattoggle between states of on and off and subsequently migrate toward asingle wall of the transport vessel. The solution is removed byaspiration and sent to a waste vessel. Low pH elution buffer (0.1 Mglycine, pH 2.0) is added and the transport vessel moves to anequilibration zone to enable elution. The transport vessel moves to apermanent magnetic field zone to allow the magnetic resin beads to mixbetween two separate and opposing magnetic fields that toggle betweenstates of on and off and subsequently migrate toward a single wall ofthe transport vessel. The solution is removed by aspiration and sent toa collection vessel. Low pH elution buffer (0.1 M glycine, pH 2.0) isadded and the transport vessel moves to an equilibration zone to enableelution. The transport vessel moves to a permanent magnetic field zoneto allow the magnetic resin beads to migrate toward a single wall of thetransport vessel. The solution is removed by aspiration and sent to acollection vessel. Regeneration buffer (0.25 M Tris; pH 8.5) is addedand the transport vessel moves to an equilibration zone to enablewashing. The transport vessel moves to a permanent magnetic field zoneto allow the magnetic resin beads to mix between two separate andopposing magnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a waste vessel.Binding/wash buffer (0.025 M Tris, 0.15 M NaCl; pH 7) is added and thetransport vessel moves to an equilibration zone to enable bufferexchange to return the magnetic resin beads to their initial conditionto complete recycling.

The affinity-purified antibody solution is transferred from thecollection vessel to the inlet of the isoelectric point-based, fluidicpurification module at a flow rate of 10 mL/min via a tubing connectionand a peristaltic pump. The solution enters a first free-flowelectrophoresis apparatus comprising an ampholyte solution designed toachieve a stable, linear pH gradient between pH 4 and pH 9 under appliedvoltage to enable operation in an isoelectric focusing mode, ade-bubbling and de-gassing system to enable continuous, long-termoperation, an active cooling system to remove Joule heat and maintain atemperature between 4° C. and 37° C., and a liquid circuit breaker toenable in-line process monitoring. This goal of this first apparatus isto separate residual host cell proteins (pI of 4-7 and 9-10) fromantibodies (pI of 7-9) into at least two fractions at the outlets of theapparatus. The outlet(s) containing the antibody fraction become(s) theinlet of a second free-flow electrophoresis apparatus connected inseries, while the outlet(s) containing host cell proteins are sent towaste collection. The antibody fraction enters the second free-flowelectrophoresis apparatus comprising two separate ampholyte solutionsand a spacer solution designed to enable operation in a highly resolvingisotachophoresis mode, a de-bubbling and de-gassing system to enablecontinuous, long-term operation, an active cooling system to removeJoule heat and maintain a temperature between 4° C. and 37° C., and aliquid circuit breaker to enable in-line process monitoring. This goalof this first apparatus is to separate residual host cell antibodies (pIof 7-9) from Etanercept (pI of 7.9) into at least two fractions at theoutlets of the apparatus. The outlet(s) containing purified Etanerceptis collected, while the outlet(s) containing antibody impurities aresent to waste collection.

The purified and isolated etanercept solution is transferred from thecollection vessel to the a high performance tangential flow filtration(HP-TFF) vessel at a flow rate of 5 mL/min via a tubing connection and aperistaltic pump to enable HP-TFF to be performed semi-continuously infed-batch mode. HP-TFF is performed to buffer exchange and furtherpurify the rituximab (diafiltration with 10 diavolumes) and thenconcentrate to enable subsequent vial filling of the retentatecontaining the purified etanercept.

This process is performed continuously for 3 months after reachingsteady-state cell culture growth conditions.

Example 2: Method of Continuous Production and Purification ofMonoclonal Antibodies Having a Dynamic Filtration Module, anAffinity-Based Purification Module, and a Free-Flow ElectrophoresisModule

Etanercept is continuously produced in a chemostat bioreactor operatingat steady-state at a titre of 4 g/L. The heterogeneous mixturecontaining etanercept, large impurities, and small impurities istransferred to a dynamic filtration module via a single output head incommunication with the input line (perfusion bioreactor bleed line) at aflow rate of 10 mL/min. Large impurities are removed by dynamicfiltration (0.45 μm PES, rolled filter membrane; mechanically smoothmembrane support structure with a parallel slotted opening andtemperature control; a wash zone; a membrane transport velocity of 1mm/sec; a vacuum gauge pressure of −0.9 bar; 2 vacuum collection vesselwith a controllable T-valve) to yield a filtrate containing etanerceptand small impurities. Once the first vacuum collection vessel reachescapacity, the flow is diverted to the second vacuum collection vesseland the first vacuum collection vessel is equilibrate to atmosphericpressure.

The filtrate is transferred from the first vacuum collection vessel(atmospheric pressure equilibrated) to the inlet of the affinity-basedpurification module at a flow rate of 10 mL/min via a tubing connectionand a peristaltic pump. Through a gasketed lid system, the filtrateenters a vessel in a carousel charged with a 40 mL dense slurry ofProtein A-coated resin beads (90 μm) suspended in a binding/wash buffer(0.025 M Tris, 0.15 M NaCl, pH 7), at the fill position of a mechanicalrotary system. Once the vessel is filled, the vessel moves to anequilibration position to allow binding for 30 minutes, while the nextvessel continues to receive the continuous filtrate flow. Following thebinding of antibodies to Protein A-coated resin beads, the vessel movesto a wash position to remove the solution containing small impurities bypressure driven flow through the basement porous membrane and direct itto waste collection. Binding/wash buffer (0.025 M Tris, 0.15 M NaCl; pH7) is added to resuspend the resin beads and enable washing for 5minutes. The wash solution is removed by pressure driven flow throughthe basement porous membrane and is directed to waste collection. Thiswash process is repeated four times to effectively wash the resin beads.Following the washing of the Protein A-coated resin beads, the vesselmoves to an elution position to elute the captured etanercept bypressure driven flow through the basement porous membrane and direct itto a collection vessel. Low pH elution buffer (0.1 M glycine; pH 2.0) isadded to resuspend the resin beads and is equilibrated for 5 minutes toenable de-binding and elution of etanercept. The eluate is removed bypressure driven flow through the basement porous membrane and isdirected to a collection vessel. This elution process is repeated fourtimes to effectively elute the etanercept from the resin beads.Following the elution of etanercept from resin beads, the vessel movesto a regeneration position to enable recycling of the resin beads.Regeneration buffer (0.25 M Tris; pH 8.5) is added to resuspend theresin beads and is equilibrated for 5 minutes to enable regeneration ofthe resin beads. This regeneration process is repeated two times toeffectively regenerate the resin beads. Binding/wash buffer (0.025 MTris, 0.15 M NaCl; pH 7) is added and is equilibrated for 5 minutes toenable buffer exchange to return the resin beads to their initialcondition to complete recycling. This buffer exchange is repeated twotimes and represents the final aspect of regeneration and recyclingprocess.

The affinity-purified antibody solution is transferred from thecollection vessel to the inlet of the isoelectric point-based, fluidicpurification module at a flow rate of 10 mL/min via a tubing connectionand a peristaltic pump. The solution enters a first free-flowelectrophoresis apparatus comprising an ampholyte solution designed toachieve a stable, linear pH gradient between pH 4 and pH 9 under appliedvoltage to enable operation in an isoelectric focusing mode, ade-bubbling and de-gassing system to enable continuous, long-termoperation, an active cooling system to remove Joule heat and maintain atemperature between 4° C. and 37° C., and a liquid circuit breaker toenable in-line process monitoring. This goal of this first apparatus isto separate residual host cell proteins (pI of 4-7 and 9-10) fromantibodies (pI of 7-9) into at least two fractions at the outlets of theapparatus. The outlet(s) containing the antibody fraction become(s) theinlet of a second free-flow electrophoresis apparatus connected inseries, while the outlet(s) containing host cell proteins are sent towaste collection. The antibody fraction enters the second free-flowelectrophoresis apparatus comprising two separate ampholyte solutionsand a spacer solution designed to enable operation in a highly resolvingisotachophoresis mode, a de-bubbling and de-gassing system to enablecontinuous, long-term operation, an active cooling system to removeJoule heat and maintain a temperature between 4° C. and 37° C., and aliquid circuit breaker to enable in-line process monitoring. This goalof this first apparatus is to separate residual host cell antibodies (pIof 7-9) from Etanercept (pI of 7.9) into at least two fractions at theoutlets of the apparatus. The outlet(s) containing purified Etanerceptis collected, while the outlet(s) containing antibody impurities aresent to waste collection.

The purified and isolated etanercept solution is transferred from thecollection vessel to the a high performance tangential flow filtration(HP-TFF) vessel at a flow rate of 5 mL/min via a tubing connection and aperistaltic pump to enable HP-TFF to be performed semi-continuously infed-batch mode. HP-TFF is performed to buffer exchange and furtherpurify the rituximab (diafiltration with 10 diavolumes) and thenconcentrate to enable subsequent vial filling of the retentatecontaining the purified etanercept.

This process is performed continuously for 3 months after reachingsteady-state cell culture growth conditions.

Example 3: Method of Continuous Production and Purification ofMonoclonal Antibodies Having a Dynamic Filtration Module, anAffinity-Based, Magnetic Purification Module, and a Charge-Based,Magnetic Purification Module

Etanercept is continuously produced in a chemostat bioreactor operatingat steady-state at a titre of 4 g/L. The heterogeneous mixturecontaining etanercept, large impurities, and small impurities istransferred to a dynamic filtration module via a single output head incommunication with the input line (chemostat bioreactor bleed line) at aflow rate of 5 mL/min. Large impurities are removed by dynamicfiltration (0.45 μm PES, rolled filter membrane; mechanically smoothmembrane support structure with a parallel slotted opening andtemperature control; a wash zone; a membrane transport velocity of 2mm/sec; a vacuum of 6 Torr; 2 vacuum collection vessel with acontrollable T-valve) to yield a filtrate containing etanercept andsmall impurities. Once the first vacuum collection vessel reachescapacity, the flow is diverted to the second vacuum collection vesseland the first vacuum collection vessel is equilibrate to atmosphericpressure.

The filtrate is transferred from the first vacuum collection vessel(atmospheric pressure equilibrated) to the inlet of the affinity-based,magnetic purification module at a flow rate of 5 mL/min via a tubingconnection and a peristaltic pump. The filtrate enters a thin-walled,transport vessel charged with 2% by weight Protein A-coated magneticresin beads (40 μm), suspended in a binding/wash buffer (0.025 M Tris,0.15 M NaCl, 0.05% Tween-20; pH 7), at the home position of a loopconveyor system. Once the transport vessel is filled, the transportvessel moves to an equilibration zone to bind for 30 minutes, while thenext transport vessel continues to receive the continuous filtrate flow.Following the binding of antibodies to Protein A-coated magnetic resinbeads, the transport vessel moves to a permanent magnetic field zone toallow the magnetic resin beads to migrate toward the wall of thetransport vessel. The solution is removed by aspiration and sent to awaste vessel. Binding/wash buffer (0.025 M Tris, 0.15 M NaCl, 0.05%Tween-20; pH 7) is added and the transport vessel moves to anequilibration zone to enable washing. The transport vessel moves to apermanent magnetic field zone to allow the magnetic resin beads to mixbetween two separate and opposing magnetic fields that toggle betweenstates of on and off and subsequently migrate toward a single wall ofthe transport vessel. The solution is removed by aspiration and sent toa waste vessel. Binding/wash buffer is added and the transport vesselmoves to an equilibration zone to enable washing. The transport vesselmoves to a permanent magnetic field zone to allow the magnetic resinbeads to mix between two separate and opposing magnetic fields thattoggle between states of on and off and subsequently migrate toward asingle wall of the transport vessel. The solution is removed byaspiration and sent to a waste vessel. Binding/wash buffer is added andthe transport vessel moves to an equilibration zone to enable washing.The transport vessel moves to a permanent magnetic field zone to allowthe magnetic resin beads to mix between two separate and opposingmagnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a waste vessel. Low pHelution buffer (0.1 M glycine, 0.05% Tween-20, pH 2.0) is added and thetransport vessel moves to an equilibration zone to enable elution. Thetransport vessel moves to a permanent magnetic field zone to allow themagnetic resin beads to mix between two separate and opposing magneticfields that toggle between states of on and off and subsequently migratetoward a single wall of the transport vessel. The solution is removed byaspiration and sent to a collection vessel. Low pH elution buffer (0.1 Mglycine, 0.05% Tween-20, pH 2.0) is added and the transport vessel movesto an equilibration zone to enable elution. The transport vessel movesto a permanent magnetic field zone to allow the magnetic resin beads tomigrate toward a single wall of the transport vessel. The solution isremoved by aspiration and sent to a collection vessel. Regenerationbuffer (0.25 M Tris, 0.05% Tween-20; pH 8.5) is added and the transportvessel moves to an equilibration zone to enable washing. The transportvessel moves to a permanent magnetic field zone to allow the magneticresin beads to mix between two separate and opposing magnetic fieldsthat toggle between states of on and off and subsequently migrate towarda single wall of the transport vessel. The solution is removed byaspiration and sent to a waste vessel. Regeneration buffer is added andthe transport vessel moves to an equilibration zone to enable magneticresin bead recycling.

The affinity-purified antibody solution is adjusted to pH 7 andtransferred from the collection vessel to the inlet of the positivecharge-based, magnetic purification module at a flow rate of 5 mL/minvia a tubing connection and a peristaltic pump. The solution enters athin-walled, transport vessel charged with 2% by weight cationicmagnetic resin beads (40 μm), suspended in an association/wash buffer(0.025 M Tris, 0.05% Tween-20, pH 7), at the home position of a loopconveyor system. Once the transport vessel is filled, the transportvessel moves to an equilibration zone to enable charge or electrostaticassociation for 30 minutes, while the next transport vessel continues toreceive the continuous affinity-purified antibody solution flow.Following the association of antibodies with the cationic magnetic resinbeads, the transport vessel moves to a permanent magnetic field zone toallow the magnetic resin beads to mix between two separate and opposingmagnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a waste vessel.Association/wash buffer (0.025 M Tris, 0.05% Tween-20, pH 7) is addedand the transport vessel moves to an equilibration zone to enablewashing. The transport vessel moves to a permanent magnetic field zoneto allow the magnetic resin beads to mix between two separate andopposing magnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a waste vessel.Association/wash buffer (0.025 M Tris, 0.05% Tween-20, pH 7) is addedand the transport vessel moves to an equilibration zone to enablewashing. The transport vessel moves to a permanent magnetic field zoneto allow the magnetic resin beads to mix between two separate andopposing magnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a waste vessel.Association/wash buffer (0.025 M Tris, 0.05% Tween-20, pH 7) is addedand the transport vessel moves to an equilibration zone to enablewashing. The transport vessel moves to a permanent magnetic field zoneto allow the magnetic resin beads to mix between two separate andopposing magnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a waste vessel.Dissociation buffer (0.1 M Tris, 0.1 M NaCl, 0.05% Tween-20, pH 7) isadded and the transport vessel moves to an equilibration zone to enabledissociation. The transport vessel moves to a permanent magnetic fieldzone to allow the magnetic resin beads to mix between two separate andopposing magnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a collection vessel.Dissociation buffer (0.025 M Tris, 0.15 M NaCl, 0.05% Tween-20, pH 7) isadded and the transport vessel moves to an equilibration zone to enabledissociation. The transport vessel moves to a permanent magnetic fieldzone to allow the magnetic resin beads to mix between two separate andopposing magnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a collection vessel.Dissociation buffer (0.025 M Tris, 0.2 M NaCl, 0.05% Tween-20, pH 7) isadded and the transport vessel moves to an equilibration zone to enabledissociation. The transport vessel moves to a permanent magnetic fieldzone to allow the magnetic resin beads to mix between two separate andopposing magnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a collection vessel.Dissociation buffer (0.025 M Tris, 0.25 M NaCl, 0.05% Tween-20, pH 7) isadded and the transport vessel moves to an equilibration zone to enabledissociation. The transport vessel moves to a permanent magnetic fieldzone to allow the magnetic resin beads to mix between two separate andopposing magnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a collection vessel.Regeneration buffer (0.025 M Tris, 0.05% Tween-20, pH 7) is added andthe transport vessel moves to an equilibration zone to enable washing.The transport vessel moves to a permanent magnetic field zone to allowthe magnetic resin beads to mix between two separate and opposingmagnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a waste vessel.Regeneration buffer (0.025 M Tris, 0.05% Tween-20, pH 7) is added andthe transport vessel moves to an equilibration zone to enable magneticresin bead recycling.

The positive charge-purified antibody solution is buffer exchanged bytangential flow filtration to 0.05 M phosphate, pH 7 and subsequentlytransferred from the collection vessel to the inlet of the negativecharge-based, magnetic purification module at a flow rate of 5 mL/minvia a tubing connection and a peristaltic pump. The solution enters athin-walled, transport vessel charged with 2% by weight anionic magneticresin beads (40 μm), suspended in an association/wash buffer (0.05 Mphosphate, 0.05% Tween-20, pH 7), at the home position of a loopconveyor system. Once the transport vessel is filled, the transportvessel moves to an equilibration zone to enable charge or electrostaticassociation for 30 minutes, while the next transport vessel continues toreceive the continuous positive charge-purified antibody solution flow.Following the association of antibodies with the anionic magnetic resinbeads, the transport vessel moves to a permanent magnetic field zone toallow the magnetic resin beads to migrate toward the wall of thetransport vessel. The solution is removed by aspiration and sent to awaste vessel. Association/wash buffer (0.05 M phosphate, 0.05% Tween-20,pH 7) is added and the transport vessel moves to an equilibration zoneto enable washing. The transport vessel moves to a permanent magneticfield zone to allow the magnetic resin beads to mix between two separateand opposing magnetic fields that toggle between states of on and offand subsequently migrate toward a single wall of the transport vessel.The solution is removed by aspiration and sent to a waste vessel.Association/wash buffer (0.05 M phosphate, 0.05% Tween-20, pH 7) isadded and the transport vessel moves to an equilibration zone to enablewashing. The transport vessel moves to a permanent magnetic field zoneto allow the magnetic resin beads to mix between two separate andopposing magnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a waste vessel.Association/wash buffer (0.05 M phosphate, 0.05% Tween-20, pH 7) isadded and the transport vessel moves to an equilibration zone to enablewashing. The transport vessel moves to a permanent magnetic field zoneto allow the magnetic resin beads to mix between two separate andopposing magnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a waste vessel.Dissociation buffer (0.05 M phosphate, 0.1 M NaCl, 0.05% Tween-20, pH 7)is added and the transport vessel moves to an equilibration zone toenable dissociation. The transport vessel moves to a permanent magneticfield zone to allow the magnetic resin beads to mix between two separateand opposing magnetic fields that toggle between states of on and offand subsequently migrate toward a single wall of the transport vessel.The solution is removed by aspiration and sent to a collection vessel.Dissociation buffer (0.05 M phosphate, 0.15 M NaCl, 0.05% Tween-20, pH7) is added and the transport vessel moves to an equilibration zone toenable dissociation. The transport vessel moves to a permanent magneticfield zone to allow the magnetic resin beads to mix between two separateand opposing magnetic fields that toggle between states of on and offand subsequently migrate toward a single wall of the transport vessel.The solution is removed by aspiration and sent to a collection vessel.Dissociation buffer (0.05 M phosphate, 0.2 M NaCl, 0.05% Tween-20, pH 7)is added and the transport vessel moves to an equilibration zone toenable dissociation. The transport vessel moves to a permanent magneticfield zone to allow the magnetic resin beads to mix between two separateand opposing magnetic fields that toggle between states of on and offand subsequently migrate toward a single wall of the transport vessel.The solution is removed by aspiration and sent to a collection vessel.Dissociation buffer (0.05 M phosphate, 0.25 M NaCl, 0.05% Tween-20, pH7) is added and the transport vessel moves to an equilibration zone toenable dissociation. The transport vessel moves to a permanent magneticfield zone to allow the magnetic resin beads to mix between two separateand opposing magnetic fields that toggle between states of on and offand subsequently migrate toward a single wall of the transport vessel.The solution is removed by aspiration and sent to a collection vessel.Regeneration buffer (0.05 M phosphate, 0.05% Tween-20, pH 7) is addedand the transport vessel moves to an equilibration zone to enablewashing. The transport vessel moves to a permanent magnetic field zoneto allow the magnetic resin beads to mix between two separate andopposing magnetic fields that toggle between states of on and off andsubsequently migrate toward a single wall of the transport vessel. Thesolution is removed by aspiration and sent to a waste vessel.Regeneration buffer (0.05 M phosphate, 0.05% Tween-20, pH 7) is addedand the transport vessel moves to an equilibration zone to enablemagnetic resin bead recycling.

The negative charge-purified and isolated etanercept solution istransferred from the collection vessel to the a high performancetangential flow filtration (HP-TFF) vessel at a flow rate of 5 mL/minvia a tubing connection and a peristaltic pump to enable HP-TFF to beperformed semi-continuously in fed-batch mode. HP-TFF is performed tobuffer exchange and further purify the rituximab (diafiltration with 10diavolumes) and then concentrate to enable subsequent vial filling ofthe retentate containing the purified etanercept.

This process is performed continuously for 3 months after reachingsteady-state cell culture growth conditions.

Example 4: Dynamic Filtration Module for Clarification of Polybeads froma Heterogeneous Mixture

The exemplary dynamic filtration module described herein provided forcontinuous dynamic filtration that successfully purified a model targetantibody, human polyclonal IgG (hIgG) from a heterogeneous mixturecomprising PolyBeads of different cell and cell debris mimicking sizes(0.5 μm, 0.75 μm, 1 μm, 2 μm, 3 μm, and 10 μm diameter at 7.3×10⁷,1.1×10⁸, 1.1×10⁸, 1.1×10⁸, 3.4×10⁷, and 1.0×10⁶ particles/mL,respectively) suspended in a 0.5 g/L solution of human polyclonal IgG(hIgG) in 1×PBS at an input flow rate of 10 mL/min from a single slotdie output head. Clarification of the PolyBeads resulted in a filtratecontaining the purified hIgG. The protein recovery at a flow rate of 10mL/min was comparable to a standard centrifugation process of 5 minutesat 10,000×g (FIGS. 11C and 11D).

The membrane support structure design and materials selection wasimportant to enable continuous membrane transport at a velocity of 0.5mm/sec while wetted and under sufficient negative pressure (gaugepressure of −0.9 bar), and thus the selection of materials that have alow static coefficient of friction when wetted for all membranecontacting surfaces (e.g. mechanically smooth PTFE) was of greatimportance (FIG. 8).

Example 5: Dynamic Filtration Module for Cell Clarification from aHeterogeneous Mixture

The exemplary dynamic filtration module described herein provided forcontinuous dynamic filtration that successfully purified a model targetantibody, human polyclonal IgG (hIgG) from a heterogeneous mixturecomprising a suspension of murine myeloma cells (2.0×10⁶ cells/mL) inRPMI media spiked with hIgG to a final concentration a 1 g/L at an inputflow rate of 2 mL/min from a single slot die output head. Clarificationof the cells and cell debris resulted in a filtrate containing thepurified hIgG. The protein recovery at a flow rate of 2 mL/min wascomparable to a standard centrifugation process of 5 minutes at 10,000×g(FIGS. 13A-13C).

The membrane support structure design and materials selection wasimportant to enable continuous membrane transport at a velocity of 0.5mm/sec while wetted and under sufficient negative pressure (gaugepressure of −0.9 bar), and thus the selection of materials that have alow static coefficient of friction when wetted for all membranecontacting surfaces (e.g. mechanically smooth PTFE) was of greatimportance (FIG. 8).

Example 6: Recovery of Proteins with Different PhysicochemicalProperties by a Dynamic Filtration Module

Dynamic filtration via the exemplary dynamic filtration module equippedwith a 0.45 μm PES filter membrane having a transport velocity of 0.5mm/sec was performed with an input flow rate of 10 mL/min for solutionsof different concentrations of of BSA (0.5-10 g/L, MW of 66,000 Da, pIof 4.5-5), a solution of Lysozyme (5 g/L, MW of 14,000 Da, pI of 11),and a solution of hIgG (0.5 g/L, MW of 150,000, pI of 6-8) and a vacuumgauge pressure of −0.9 bar to evaluate protein recovery, as determinedby spectrophotometric analysis of the resulting filtrates (n=3 for eachsolution) by BCA assay. The protein recovery was similar for allproteins and was observed to be >96% (FIG. 12A).

Example 7: Effect of Filter Membrane Material on Dynamic FiltrationModule Performance

Dynamic filtration via the exemplary dynamic filtration module equippedwith different low protein binding filter membrane materials (PES,hydrophilic PVDF) and pore sizes (0.45 μm, 0.22 μm) having a transportvelocity of 0.5 mm/sec was performed with an input flow rate of 10mL/min for solutions of hIgG (0.5 g/L) and a vacuum gauge pressure of−0.9 bar to evaluate protein recovery, as determined byspectrophotometric analysis of the resulting filtrates (n=3 for eachsolution) by BCA assay. The protein recovery was similar for each filtermembrane material and pore size and was observed to be >96% (FIG. 12B).

Example 8: Effect of Different Membrane Support Structure Geometries andMaterials on Dynamic Filtration Module Performance

Dynamic filtration via the exemplary dynamic filtration module equippedwith mechanically smooth PTFE membrane support structures havingdifferent opening geometries (5 parallel slots, porous hydrophilic PEinsert) and a 0.45 μm PES filter membrane having a transport velocity of0.5 mm/sec was performed with an input flow rate of 10 mL/min forsolutions of hIgG (0.5 g/L) and a vacuum gauge pressure of −0.9 bar toevaluate protein recovery, as determined by spectrophotometric analysisof the resulting filtrates (n=3 for each solution) by BCA assay. Theprotein recovery was similar for each membrane support structure and wasobserved to be >96% (FIG. 12C).

Example 9: Continuous, Long-Term Dynamic Filtration Module Performance

Continuous dynamic filtration via the exemplary dynamic filtrationmodule equipped with mechanically smooth PTFE membrane support structureand a 0.45 μm PES filter membrane having a transport velocity of 0.5mm/sec was performed with input flow rates of either 5 or 10 mL/min forsolutions of Lysozyme (0.5 g/L) and a vacuum gauge pressure of −0.9 barto evaluate longitudinal protein recovery over a 25 minute duration, asdetermined by spectrophotometric analysis of the resulting filtrates byBCA assay. The protein recovery was similar for each membrane supportstructure and was observed to be >96% (FIG. 12D).

Example 10: Affinity-Based, Magnetic Purification of Polyclonal HumanIgg from a Mixture

The exemplary affinity-based, magnetic purification module describedherein was utilized to purify hIgG from a mixture containing 2 g/L hIgG(affinity target) and 1 g/L Lysozyme (small impurity). Four millilitersof the mixture containing 8 mg hIgG and 4 mg Lysozyme in binding/washbuffer (0.025 M Tris, 0.15 M NaCl; pH 7) were added at 10 mL/min to athin-walled vessel charged with 500 μL of settled, high affinity ProteinA/G magnetic agarose (dynamic binding capacity of >40 mg hIgG/mL settledresin) and equilibrated for 30 minutes with gentle mixing to enablebinding. Following the 30 minute binding equilibration, a permanent Ndmagnet was manually placed within close proximity to the thin-walledvessel (e.g. mimicking a pick and place robotics system) to attract themagnet affinity beads to the vessel wall and allow for collection of thesolution containing unbound hIgG and small impurities by aspiration.Following aspiration, the vessel was filled with 4 mL of binding/washbuffer (0.025 M Tris, 0.15 M NaCl; pH 7) at 10 mL/min and equilibratedfor 5 minutes with gentle mixing to wash. Following the 5 minute wash, apermanent Nd magnet was manually placed within close proximity to thethin-walled vessel (e.g. mimicking a pick and place robotics system) toattract the magnet affinity beads to the vessel wall and allow forcollection of the wash solution by aspiration. The wash step wasrepeated for a total 3 washes. Following aspiration of the washfractions, the vessel was filled with 4 mL of low pH elution buffer (0.1M glycine; pH 2) at 10 mL/min and equilibrated for 10 minutes withgentle mixing to elute. Following the 10 minute elution, a permanent Ndmagnet was manually placed within close proximity to the thin-walledvessel (e.g. mimicking a pick and place robotics system) to attract themagnet affinity beads to the vessel wall and allow for collection of theeluate fraction by aspiration. The elution step was repeated for a totalof 3 elutions. Following collection of the 3 eluate fractions, thevessel was filled with 4 mL of low pH elution buffer (0.1 M glycine; pH2) at 10 mL/min and equilibrated for 5 minutes with gentle mixing tocompletely remove any residually bound hIgG to initiate regeneration ofthe magnetic affinity beads. Following the 5 minute residual elution, apermanent Nd magnet was manually placed within close proximity to thethin-walled vessel (e.g. mimicking a pick and place robotics system) toattract the magnet affinity beads to the vessel wall and allow forcollection of the first regeneration solution by aspiration. Followingcollection of the first regeneration solution, the vessel was filledwith 4 mL of regeneration buffer (0.25 M Tris; pH 8.5) at 10 mL/min andequilibrated for 5 minutes with gentle mixing to neutralize the pH ofthe magnetic affinity beads and remove any residual hIgG and smallimpurities to regenerate the magnetic affinity beads. Following the 5minute regeneration, a permanent Nd magnet was manually placed withinclose proximity to the thin-walled vessel (e.g. mimicking a pick andplace robotics system) to attract the magnet affinity beads to thevessel wall and allow for collection of the second regeneration solutionby aspiration. Following collection of the second regeneration solution,the vessel was filled with 4 mL of a second regeneration buffer (0.025 MTris, 0.15 M NaCl; pH 7) at 10 mL/min and equilibrated for 5 minuteswith gentle mixing to buffer exchange the magnetic affinity beads andreturn the magnetic affinity beads to their initial condition. Followingthe 5 minute regeneration, a permanent Nd magnet was manually placedwithin close proximity to the thin-walled vessel (e.g. mimicking a pickand place robotics system) to attract the magnet affinity beads to thevessel wall and allow for collection of the buffer exchange solution byaspiration. The buffer exchange step was repeated for a total of 2 timesto enable reuse of the magnetic affinity beads. The collected fractionsfor 3 consecutive process cycles and magnetic affinity bead recyclingwere analyzed spectrophotometrically by BCA and were observed to berobust and reproducible (FIG. 20A). The collected fractions for the 3consecutive process cycles and magnetic affinity bead recycling werefurther characterized by SDS-PAGE to confirm the reproducibility andshow the ability to purify the hIgG (FIG. 20B).

Example 11: Affinity-Based Purification of Polyclonal Human Igg from aMixture

The exemplary affinity-based purification module described herein wasutilized to purify hIgG from a mixture containing 2 g/L hIgG (affinitytarget) and 1 g/L Lysozyme (small impurity). Four milliliters of themixture containing 8 mg hIgG and 4 mg Lysozyme in binding/wash buffer(0.025 M Tris, 0.15 M NaCl; pH 7) were added at 10 mL/min via with a lidsystem to a vessel containing a basement glass frit and charged with 500μL of settled, high affinity Protein A agarose (90 μm, dynamic bindingcapacity of >35 mg hIgG/mL settled resin) and equilibrated for 30minutes with gentle mixing to enable binding. Following the 30 minutebinding equilibration, compressed air was introduced to the vessel atabout 1 psi to allow for collection of the solution containing unboundhIgG and small impurities by pressure driven flow through. Followingcollection, the vessel was filled with 4 mL of binding/wash buffer(0.025 M Tris, 0.15 M NaCl; pH 7) at 10 mL/min and equilibrated for 5minutes with gentle mixing to wash. Following the 5 minute wash,compressed air was introduced to the vessel at about 1 psi to allow forcollection of the wash solution by pressure driven flow through. Thewash step was repeated for a total 3 washes. Following collection of the3 wash fractions, the vessel was filled with 4 mL of low pH elutionbuffer (0.1 M glycine; pH 2) at 10 mL/min and equilibrated for 10minutes with gentle mixing to elute. Following the 10 minute elution,compressed air was introduced to the vessel at about 1 psi to allow forcollection of the eluate fraction by pressure driven flow through. Theelution step was repeated for a total of 3 elutions. Followingcollection of the 3 eluate fractions, the vessel was filled with 4 mL oflow pH elution buffer (0.1 M glycine; pH 2) at 10 mL/min andequilibrated for 5 minutes with gentle mixing to completely remove anyresidually bound hIgG to initiate regeneration of the affinity resinbeads. Following the 5 minute residual elution, compressed air wasintroduced to the vessel at about 1 psi to allow for collection of thefirst regeneration solution. Following collection of the firstregeneration solution, the vessel was filled with 4 mL of regenerationbuffer (0.25 M Tris; pH 8.5) at 10 mL/min and equilibrated for 5 minuteswith gentle mixing to neutralize the pH of the affinity resin beads andremove any residual hIgG and small impurities to regenerate the affinityresin beads. Following the 5 minute regeneration, compressed air wasintroduced to the vessel at about 1 psi to allow for collection of thesecond regeneration solution. Following collection of the secondregeneration solution, the vessel was filled with 4 mL of a secondregeneration buffer (0.025 M Tris, 0.15 M NaCl; pH 7) at 10 mL/min andequilibrated for 5 minutes with gentle mixing to buffer exchange theaffinity resin beads and return the affinity resin beads to theirinitial condition. Following the 5 minute regeneration, compressed airwas introduced to the vessel at about 1 psi to allow for collection ofthe first regeneration solution. The buffer exchange step was repeatedfor a total of 2 times to enable reuse of the affinity resin beads. Thecollected fractions for 3 consecutive process cycles and magneticaffinity bead recycling were analyzed spectrophotometrically by BCA andwere observed to be robust and reproducible (FIG. 27A). The collectedfractions for the 3 consecutive process cycles and magnetic affinitybead recycling were further characterized by SDS-PAGE to confirm thereproducibility and show the ability to purify the hIgG (FIG. 27B).

Example 12: Separation of Small-Molecules by High Flow Rate, IsoelectricFocusing Free-Flow Electrophoresis Using an Isoelectric Point-Based,Fluidic Purification Module

A mixture of Rhodamine 6G (0.25 mg/mL) and Fluorescein (0.25 mg/mL) wasintroduced to the central inlet (inlet 3) of an exemplary free-flowelectrophoresis apparatus comprising an anodic channel (H₂SO₄), acathodic channel (NaOH), and a main separation channel having fiveinlets and five outlets flowing an ampholyte solution designed toachieve a stable, linear pH gradient between pH 2 and pH 12 underapplied voltage to enable operation in an isoelectric focusing mode, ade-bubbling and de-gassing system to enable continuous, long-termoperation, and an active cooling system (a thermal chuck with chilledcirculating ethylene glycol/water) to remove Joule heat through thebottom plate and maintain a temperature between 4° C. and 37° C. When novoltage was applied, the mixture followed laminar flow and exited theapparatus at the central outlet (outlet 3) (FIGS. 40A and 40B). When1000V is applied across the main separation channel having an ampholyteand sample input flow rate of 10 mL/min, a linear pH gradient wasestablished and Rhodamine 6G and Fluorescein migrated to the cathode andanode, respectively, consistent with theoretical electrophoreticmobility predictions (FIGS. 40C and 40D). Spectrophotometric analysis ofthe fractions collected from outlet 2 and outlet 4 showed the presenceof purified Rhodamine 6G and purified Fluorescein, respectively (FIG.40E).

Example 13: Separation of Small-Molecules by High Flow Rate,Isotachophoresis Using an Isoelectric Point-Based, Fluidic PurificationModule

A mixture of Rhodamine 6G (0.25 mg/mL) and Fluorescein (0.25 mg/mL) wasintroduced to the central inlet (inlet 3) of an exemplary free-flowelectrophoresis apparatus comprising an anodic channel (H₂SO₄), acathodic channel (NaOH), and a main separation channel having fiveinlets and five outlets flowing a basic ampholyte solution (inlets 1 and2), a spacer solution (inlet 3), and an acidic ampholyte solution(inlets 4 and 5) to enable operation in an isoelectric focusing mode, ade-bubbling and de-gassing system to enable continuous, long-termoperation, and an active cooling system (a thermal chuck with chilledcirculating ethylene glycol/water) to remove Joule heat through thebottom plate and maintain a temperature between 4° C. and 37° C. When novoltage was applied, the mixture followed laminar flow and exited theapparatus at the central outlet (outlet 3). When 250V was applied acrossthe main separation channel having an ampholyte and sample input flowrate of 5 mL/min, Rhodamine 6G and Fluorescein migrated to the cathodeand anode, respectively, forming highly focused, highly concentrated,and highly resolved bands (FIGS. 44A and 44B).

Example 14: Separation of Basic and Acidic Small-Molecules by High FlowRate, Isoelectric Focusing Free-Flow Electrophoresis Using anIsoelectric Point-Based, Fluidic Purification Module

mixtures of Basic Fuchsin (0.05 mg/mL) and Fluorescein (0.25 mg/mL) orCrystal Violet (0.05 mg/mL) and Fluorescein (0.25 mg/mL) were introducedto the central inlet (inlet 3) of an exemplary free-flow electrophoresisapparatus comprising an anodic channel (H₂SO₄), a cathodic channel(NaOH), and a main separation channel having five inlets and fiveoutlets flowing an ampholyte solution designed to achieve a stable,linear pH gradient between pH 2 and pH 12 under applied voltage toenable operation in an isoelectric focusing mode, a de-bubbling andde-gassing system to enable continuous, long-term operation, and anactive cooling system (a thermal chuck with chilled circulating ethyleneglycol/water) to remove Joule heat through the bottom plate and maintaina temperature between 4° C. and 37° C. When no voltage was applied, themixtures followed laminar flow and exited the apparatus at the centraloutlet (outlet 3). When 500V was applied across the main separationchannel having an ampholyte and sample input flow rate of 5 mL/min, alinear pH gradient was established and Basic Fuchsin and Fluoresceinmigrated to the cathode and anode, respectively, consistent withtheoretical electrophoretic mobility predictions (FIG. 42A). Similarly,when 500V was applied across the main separation channel having anampholyte and sample input flow rate of 5 mL/min, a linear pH gradientwas established and Crystal Violet and Fluorescein migrated to thecathode and anode, respectively, consistent with theoreticalelectrophoretic mobility predictions (FIG. 42B).

Example 15: Effect of Increasing E-Field on the Separation of Basic andAcidic Small-Molecules by High Flow Rate, Isoelectric Focusing Free-FlowElectrophoresis Using an Isoelectric Point-Based, Fluidic PurificationModule

A mixture of Basic Fuchsin (0.005 mg/mL) and Fluorescein (0.25 mg/mL)was introduced to the central inlet (inlet 3) of an exemplary free-flowelectrophoresis apparatus comprising a main separation channel havingfive inlets and ten outlets flowing an ampholyte solution designed toachieve a stable, linear pH gradient between pH 2 and pH 12 underapplied voltage to enable operation in an isoelectric focusing mode, ananodic and cathodic channel flowing the same ampholyte as the separationchannel, a de-bubbling and de-gassing system to enable continuous,long-term operation, a liquid circuit breaker, and an active coolingsystem (a thermal chuck with chilled circulating ethylene glycol/water)to remove Joule heat through the bottom plate and maintain a temperaturebetween 4° C. and 37° C. When no voltage was applied, the mixturefollowed laminar flow and exited the apparatus at the central outlets(outlets 4 and 5) (FIG. 43A). When voltage was applied across the mainseparation channel having an ampholyte and sample input flow rate of 5mL/min, a linear pH gradient was established and Basic Fuchsin andFluorescein migrated to the cathode and anode, respectively, consistentwith theoretical electrophoretic mobility predictions (FIGS. 43B-43D).As the applied voltage was increased from 600V (FIG. 43B), to 900V (FIG.43C) to 1100V (FIG. 43D) to generate an increase in the E-fieldstrength, the separation of the two molecules was observed toproportionally increase over the length of the main separation channel.

Example 16: Separation of Acidic and Basic Proteins by High Flow Rate,Isoelectric Focusing Free-Flow Electrophoresis Using an IsoelectricPoint-Based, Fluidic Purification Module

A mixture of BSA (0.5 mg/mL, pI of 4-5) and Lysozyme (0.25 mg/mL, pI of11) was introduced to the central inlet (inlet 3) of an exemplaryfree-flow electrophoresis apparatus comprising an anodic channel(H₂SO₄), a cathodic channel (NaOH), and a main separation channel havingfive inlets and five outlets flowing an ampholyte solution designed toachieve a stable, linear pH gradient between pH 2 and pH 12 underapplied voltage to enable operation in an isoelectric focusing mode, ade-bubbling and de-gassing system to enable continuous, long-termoperation, and an active cooling system (a thermal chuck with chilledcirculating ethylene glycol/water) to remove Joule heat through thebottom plate and maintain a temperature between 4° C. and 37° C. When novoltage was applied, the mixture followed laminar flow and exited theapparatus at the central outlet (outlet 3) (FIGS. 45A and 45B). When850V was applied across the main separation channel having an ampholyteand sample input flow rate of 10 mL/min, a linear pH gradient wasestablished and Lysozyme and BSA migrated to the cathode and anode(FIGS. 45C and 45D), respectively, consistent with theoreticalelectrophoretic mobility predictions (FIG. 45E).

Example 17: Separation of Human Polyclonal IGG by High Flow Rate,Isoelectric Focusing Free-Flow Electrophoresis Using an IsoelectricPoint-Based, Fluidic Purification Module

A mixture of hIgG (0.5 mg/mL, pI of 6-8) and Lysozyme (0.25 mg/mL, pI of11) was introduced to the central inlet (inlet 3) of an exemplaryfree-flow electrophoresis apparatus comprising an anodic channel(H₂SO₄), a cathodic channel (NaOH), and a main separation channel havingfive inlets and five outlets flowing an ampholyte solution designed toachieve a stable, linear pH gradient between pH 2 and pH 12 underapplied voltage to enable operation in an isoelectric focusing mode, ade-bubbling and de-gassing system to enable continuous, long-termoperation, and an active cooling system (a thermal chuck with chilledcirculating ethylene glycol/water) to remove Joule heat through thebottom plate and maintain a temperature between 4° C. and 37° C. When novoltage was applied, the mixture followed laminar flow and exited theapparatus at the central outlet (outlet 3) (FIGS. 46A and 46C). When1000V was applied across the main separation channel having an ampholyteand sample input flow rate of 5 mL/min, a linear pH gradient wasestablished and the Lysozyme was observed to migrate to the cathode(FIGS. 46A and 46C), consistent with theoretical electrophoreticmobility predictions (FIG. 46B). Increasing the applied voltage to 1500Vresulted in an increase in migration of the Lysozyme to the cathode.This increase in applied voltage also resulted in the migration of thehIgG to the cathode and anode (FIGS. 46A and 46C), consistent withtheoretical electrophoretic mobility predictions for the range of pIsinherent to a polyclonal antibody (FIG. 46B).

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. Allreferences, e.g., U.S. patents, U.S. patent application publications,PCT patent applications designating the U.S., published foreign patentsand patent applications cited herein are incorporated herein byreference in their entireties. Genbank and NCBI submissions indicated byaccession number cited herein are incorporated herein by reference. Allother published references, documents, manuscripts, and scientificliterature cited herein are incorporated herein by reference. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A free-flow electrophoresis apparatus forseparating a mixture into two or more fractions, at least one fractioncontaining a biological product, comprising: at least one inlet and atleast one outlet configured to permit continuous fluid flow between theat least one inlet and the at least one outlet; at least one fluidicchannel created between two parallel plates and configured to create anelectric field gradient orthogonal to the direction of fluid flow;electrode channels comprising an anodic electrode channel and a cathodicelectrode channel, wherein the electrode channels are configured to beconnected to the main separation channel by liquid contact through awall gap positioned between the electrode channels and the mainseparation channel; at least one electrode channel de-bubbler comprisingat least one gas permeable and hydrophobic membrane or porous materialconfigured to remove electrolysis bubbles near the point of generationby a vacuum system to create a bubble-free main separation channel; atleast one liquid circuit breaker configured to disconnect a liquidconnected to voltage prior to interacting with at least one sensor ordetector; an active cooling system; and at least one collection vessel.2. The apparatus of claim 1, wherein a top portion of the electrodechannels are sealed with at least one gas permeable and hydrophobicmembrane in communication with a vacuum system to remove bubbles, andwherein the electrode channels are open at the bottom of the channelsand configured to enable liquid contact of electrode solution with themain separation channel solution through a wall gap.
 3. The apparatus ofclaim 1, wherein wall gap is about 0.01 mm to about 0.25 mm.
 4. Theapparatus of claim 1, wherein the liquid circuit breaker comprises apressurized vessel configured to maintain flow rate and creates dropletsthat break the circuit from the solution connected to voltage.
 5. Theapparatus of claim 1, wherein the at least one sensor is an in-linesensor.
 6. The apparatus of claim 1, further comprising at least twofree-flow electrophoresis apparatuses connected in series and operatedin an isoelectric focusing mode, a zone electrophoresis mode, anisotachophoresis mode, or combinations thereof, to enable a stagedpurification.